Negative serum (1,3) -ß-D-glucan has a low power to exclude Pneumocystis jirovecii pneumonia (PJP) in HIV-uninfected patients with positive qPCR.

OBJECTIVE: The current study evaluated the diagnostic performance of serum (1,3)-beta-D Glucan (BDG) in differentiating PJP from P. jirovecii-colonization in HIV-uninfected patients with P. jirovecii PCR-positive results. METHODS: This was a single-center retrospective study between 2019 and 2021. The diagnosis of PJP was based on the following criteria: detection of P. jirovecii in sputum or BAL specimen by qPCR or microscopy; Meet at least two of the three criteria: (1) have respiratory symptoms of cough and/or dyspnea, hypoxia; (2) typical radiological picture findings; (3) receiving a complete PJP treatment. After exclusion, the participants were divided into derivation and validation cohorts. The derivation cohort defined the cut-off value of serum BDG. Then, it was verified using the validation cohort. RESULTS: Two hundred and thirteen HIV-uninfected patients were enrolled, with 159 PJP and 54 P. jirovecii-colonized patients. BDG had outstanding specificity, LR, and PPV for PJP in both the derivation (90.00%, 8.900, and 96.43%) and the validation (91.67%, 9.176, and 96.30%) cohorts at ≥ 117.7 pg/mL. However, it had lower sensitivity and NPV in the derivation cohort (89.01% and 72.97%), which was even lower in the validation cohort (76.47% and 57.89%). Of note, BDG ≥ 117.7 pg/mL has insufficient diagnostic efficacy for PJP in patients with lung cancer, interstitial lung disease (ILD) and nephrotic syndrome. And although lymphocytes, B cells, and CD4+ T cells in PJP patients were significantly lower than those in P. jirovecii-colonized patients, the number and proportion of peripheral blood lymphocytes did not affect the diagnostic efficacy of serum BDG. CONCLUSIONS: Serum BDG ≥ 117.7 pg/mL could effectively distinguish P. jirovecii-colonization from infection in qPCR-positive HIV-uninfected patients with infectious diseases, solid tumors (excluding lung cancer), autoimmune or inflammatory disorders, and hematological malignancies. Of note, for patients with lung cancer, ILD, and nephrotic diseases, PJP should be cautiously excluded at BDG < 117.7 pg/mL.

Prevalence and Genotype Distribution of Human Papillomavirus Among Healthy Females in Beijing, China, 2016-2019.

OBJECTIVE: Human papillomavirus (HPV) infection, especially with high-risk HPV (HR-HPV) genotypes, is closely associated with cervical cancer. This study aimed to observe the epidemiological characteristics of HPV infection among healthy women in Beijing, China. MATERIALS AND METHODS: Cervical specimens were collected from 29,436 healthy women, who underwent health check-ups in Peking Union Medical College Hospital between 2016 and 2019. A commercial kit was used for the detection of 15 HR-HPV and two low-risk HPV (LR-HPV) genotypes. RESULTS: A total of 3586 (12.18%) participants tested positive for HPV, 3467 of which were infected with HR-HPVs. The most prevalent genotypes were HPV52, 58, 16, 51, and 56. Moreover, while infection with a single genotype (9.84%) was more prevalent, HPV16+52 was the most common combination in those infected with multiple HPVs. Furthermore, the highest infection rate among age groups was in women aged <25 years (20.92%). No significant difference in the prevalence was observed from 2016 to 2019. However, HPV incidence in Beijing was significantly different than that in all other areas in China, except for Zhengzhou (p < 0.05). CONCLUSION: Our findings could serve as potential reference for better understanding of the epidemiological characteristics of HPV infection in Beijing.

[Infection Status of Human Papilloma Virus,Ureaplasma Urealyticum, Chlamydia Trachomatis,and Neisseria Gonorrhoeae].

Objective To analyze the infection status of human papilloma virus (HPV),Ureaplasma urealyticum (UU),Chlamydia trachomatis (CT),and Neisseria gonorrhoeae (NG) in clinical patients.Methods The laboratory specimens including urine,urethral swabs,and cervical swabs from 870 patients from January 1st 2014 to December 31st 2017 were retrospectively analyzed. HPV-DNA was detected by multiplex fluorescent PCR,and the UU-RNA,CT-RNA,and NG-RNA were determined by isothermal nucleic acid amplification. The positive rate of each pathogen and the distribution of positive rate between male and female patients were calculated. The samples were further divided into HPV-positive group and HPV-negative group,and the positive rates of UU-RNA,CT-RNA,and NG-RNA in these two groups were compared.Results The highest positive rate was 53.68%(467/870) for UU-RNA,followed by HPV-DNA [32.41%(282/870) ]and NG-RNA [2.18%(19/870)]. The total positive rate of high-risk (HR)-HPV(subtypes:16,18,31,33,35,39,45,51,52,56,58,59,and 68) [31.52%(209/663)]and UU in female patients [60.93%(404/663)] was significantly higher than that in male patients [17.39%(36/207),30.34%(63/207)](both P<0.001). The male patients had significantly higher CT positive rate in HR-HPV-positive group than in HR-HPV-negative group [22.58%(7/31) vs. 4.54%(8/176)](P<0.001). The female patients had significantly higher CT positive rate in HR-HPV-positive group than in HR-HPV-negative group [10.5%(21/200) vs. 5.61%(26/463)](P=0.024). The UU-RNA positive rate of females in the low-risk (LR)-HPV (subtypes:6 and 11) positive group was significantly higher than that in LR-HPV negative group [70.83%(34/48) vs.2.11%(13/615)](P<0.001).Conclusions Women are more susceptible to HR-HPV and UU infections. HR-HPV-positive patients are more likely to experience CT infection. In contrast,co-infection with UU is more common in LR-HPV-positive females.

Infection Modes and Subtypes of Human Papilloma Virus in Patients.

Objective To investigate the prevalence of human papilloma virus(HPV)subtypes in patients and to provide an evidence for the prevention and treatment of HPV infection and the development of HPV vaccine. Methods Multiplex PCR was used to detect HPV DNA in 6917 patients in Peking Union Medical College Hospital from January 1,2013 to June 30,2015.Totally 5586 patients entered the final analysis after the repeat samples were deleted.The total positive rate of HPV subtypes(including high-risk subtypes including HPV-16,18,31,33,35,39,45,51,52,56,58,59,and 68 and low-risk subtypes including HPV-6 and 11)and the infection status of different age were analyzed. Results The total positive rate of HPV was 36.29%(2027/5586).The positive rate of high-risk subtype was 24.92%(1392/5586)and low-risk subtype was 1.66%(93/5586).The positive rate of multiple was 9.70%(542/5586)and multiple high-risk subtype was 7.75%(433/5586).The positive rate of high-risk subtype and multiple were 25.52%(1366/5353)and 11.16%(26/233)in female and 9.99%(535/5353)and 3.00%(7/233)in male,there were significantly difference(χ2=24.61,χ2=12.45,all P<0.001).The positive rate of low-risk subtypes(3.86%,9/233)in males was significantly higher than that in females(1.57%,84/5353)(χ2=5.84,P=0.007).The high-risk HPV subtype infection mainly was seen in patients aged 31-50 years and the low-risk HPV subtype infection mainly in patients aged 21-40 years.The age of multiple HPV infections from 31-40 years.The lowest turn negative rates of subtype were HPV52 and HPV58.The top three HPV subtypes with the highest positive rates were HPV52,HPV16,and HPV58.Conclusions The positive rates of HPV type are different between male and female patients.The males are mainly infected with low-risk subtypes,whereas the females with high-risk subtypes and the multiple HPV subtypes.The top three high-risk subtypes are HPV-52,16,and 58.HPV subtypes with the lowest secondary negative rates are HPV-52 and 58.HPV infection is mainly seen in young individuals.

Efficient replication of blood-borne hepatitis C virus in human fetal liver stem cells.

The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION: Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).

A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

[Value of detecting epidermal growth factor receptor mutations in non-small cell lung cancer tissue by TaqMan-amplification refractory mutation system].

OBJECTIVE: To explore the value of detecting mutations on epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) tissue by TaqMan-amplification refractory mutation system (TaqMan-ARMS). METHODS: TaqMan-ARMS and DNA sequencing were used to detect the EGFR exon 19 and 21 mutations in tumor tissues and the samples collected from 199 patients at 4 different 3A hospitals in Beijing from January 2008 to March 2011. RESULTS: The rate of mutations in EGFR exon 19 and 21 was 19.1% (38/199), according to their different pathological types. Based upon TaqMan-ARMS, the classification was as followed: adenocarcinoma (35.0% (36/103)), squamous carcinoma (2.2% (2/93)) and adenosquamous carcinoma (0). According to DNA sequencing, they were 19.6% (39/199), 35.9% (37/103), 2.2% (2/93) and 0 respectively. Thus, no statistically significant difference existed between two methods (McNemar Test, P = 1.000, κ = 0.984). The mutation rate of adenocarcinoma was higher than those of squamous and adenosquamous carcinoma. CONCLUSION: The detection of EGFR mutations is highly consistent in the NSCLC tissue by the methods of TaqMan-ARMS and DNA sequencing.

[Analysis of cytomegaloviral antigenemia test and re-test after antiviral chemotherapy in patients with autoimmune and non-autoimmune diseases].

OBJECTIVE: To retrospectively analyze the cytomegalovirus (CMV) antigenemia test and re-test after antiviral chemotherapy in patients with autoimmune and non-autoimmune diseases. METHODS: CMV Brite kit and indirect immunofluorescence were used to detect CMVpp65 antigenemia in 6471 peripheral blood leukocyte specimens from 5325 clinic and hospitalized patients with clinically suspicious CMV infections from May 2008 to February 2012. And the positive results were defined as episodes of systemic CMV activity. RESULTS: In 6471 EDTA-treated peripheral blood specimens, 948 (14.6%) were found with positive CMV antigenemia. The average positive rate from 13 kinds of autoimmune diseases was 34.9% (670/1922) in which systemic lupus erythematosus patients had the highest (52.4%, 551/1052). Meanwhile, the average positive rate from 12 kinds of non-autoimmune diseases was only 6.1% (144/2367) in which it was 17.3% (27/156) in patients with respiratory/acute renal failure, acquired immunodeficiency syndrome (AIDS) and kidney transplant recipients. And 189 patients with positive antigenemia were re-tested after antiviral chemotherapy and only 64 (33.9%) were converted negatively. CONCLUSIONS: Patients with autoimmune diseases have replaced traditionally immunocompromised patients, e.g. AIDS and kidney transplant recipient, to become the highest risk group of systemic CMV activity. Negative conversion rate of CMV antigenemia is low after antiviral chemotherapy.

[Analysis of the correlation between PreS1-Ag, PreS2-Ag, HBeAg and HBV DNA in the serum of chronic hepatitis B patients].

OBJECTIVE: To investigate the correlation between the serum indices and HBV DNA. METHODS: 100 chronic HBV patients and 40 healthy controls were enrolled in this study. The patients have not received any treatment with interferon (IFN) and similar nucleotide, and featured with normal ALT/AST, positive HBV DNA examined by using internal-quantitative standard PCR. Serum indices were measured by micro-particle enzyme immunoassay analysis and ELISA. RESULTS: In chronic HBV patients with positive HBV DNA, the positive percentage were 75% for HBeAg, 25% for anti-HBe, 69% for PreS1-Ag, and 77% for PreS2-Ag. In 75 HBeAg positive cases, there were 54 cases of PreS1-Ag positive and 60 cases of PreS2-Ag positive; while among anti-HBe positive cases, there were 14 cases of PreS1-Ag positive and 17 cases of PreS2-Ag positive. There was no significant difference of the positive percentage of PreS1-Ag and PreS2-Ag between HBeAg positive and anti-HBe positive (both P > 0.05). Under circumstance of HBeAg positive, the percentage of both PreS1-Ag and PreS2-Ag negative (8%) was significant lower than that of anti-HBe positive (28%) (P < 0.05). There were 72 cases with consistent positive or negative for both PreS1-Ag and PreS2-Ag. CONCLUSION: For hepatitis B patients with positive HBV DNA, the differences of positive percentages for HBeAg, PreS1-Ag and PreS2-Ag are moderate. The positive percentages of PreS1-Ag and PreS2-Ag do not correlate with HBeAg positive. The negative percentages of PreS1-Ag and PreS2-Ag correlate with anti-HBe positive.

[Study on the correlation among quantification of HBV-DNA and HBeAg, anti-HBe in hepatitis B carriers].

OBJECTIVE: To better understand the duplication of hepatitis B virus (HBV) in order to improve clinical diagnoses and treatments via quantitative measurement of HBV-DNA and comparison of correlation of HBV-DNA with HBeAg and anti-HBe. METHODS: For 883 hepatitis B patients with positive HBsAg, HBV-DNA was measured by COBAS AMPLICOR HBV MONITOR reagent and COBAS AMPLICOR quantitative PCR instrument. Microparticle enzyme immunoassay analysis (MEIA) was then carried out with fully automatic enzyme immunoassay analysis instrument made by Abbott Axsym from the U.S. to measure HBeAg and anti-HBe. Correlation was analysed by SPSS. RESULTS: (1)Positive correlation between 690 HBV-DNA positive and HBeAg positive with r= 0. 505 (P< 0.01) was found with mean values as:HBV-DNA:7.12 x 10(12) copies/ml;HBeAg:218.31 S/CO. HBV-DNA:10(4) copies/ml, HBeAg: 104 S/CO; HBV-DNA: 10(5)-10(8) copies/ml, HBeAg: 112 S/CO; HBV-DNA: 10(9)-10(15) copies/ml, HBeAg: 252 S/CO. (2) No correlation was found between 193 HBV-DNA and anti-HBe + with r= -0.052(P= 0.477> 0.05) with Mean: HBV-DNA: 8.0x 10(10) copies/ml anti-HBe: 0.18 S/CO. CONCLUSION: HBV-DNA and HBeAg appeared to have had linear correlation, showing that HBeAg> 100 S/CO,HBV-DNA> 10(4) copies/ml and hepatitis B virus were reproduced. However, HBV-DNA did not show linear correlation with anti-HBe as HBeAg negative and anti-HBe positive, the level of hepatitis B viral replication decrease slightly. But the virus load is still high. Infectivity can not neglect.

[Correlation of hepatitis B e antigen with hepatitis B virus DNA in the serum of chronic hepatitis B patients after treatment].

OBJECTIVE: To analyze the correlation of hepatitis B virus (HBV) DNA with the serological markers of HB in the serum of chronic HB patients after treatment PCR method and to analyze the status of these markers and the multiplication of virus. METHODS: Peripheral blood samples were collected from 480 chronic HB patients, aged 15 - 50, who had been treated by anti-nucleotide drugs or traditional Chinese herbs and showed normal ALT/AST. Both COBAS AMPLICOR HBV MONIORTM kit (internal-standard PCR method) and Light Cycler real time fluorescent quantitative PCR instrument (external-quantitative standard PCR method) were used to measure the HBV DNAS level. 42 of the 312 patients with the HBV DNA level lower than the minimum test limit measured by COBAS AMPLICOR HBV MONIORTM kit and HBeAg positive (>4 S/CO) underwent microparticle enzyme immunoassay (MEIA) to test the HBsAg, anti-HBs, HBeAg, anti-HBe, and HBcIgG. RESULTS: Seven of the 42 patients with HBV DNA negative measured by COBAS AMPLICOR HBV MONIORTM kit lower then the minimum test limit were shown as HBV DNA positive by Light Cycler real time fluorescent quantitative PCR. The 42 patients were HBsAg (+), anti-HBs (-), HBeAg (+), anti-HBe (-), and anti-HBcIgG (-), with an average HBeAg level of 42.26 S/CO and a positive HBeAg rate of 13.46%. CONCLUSION: HBeAg positivity does not necessarily means an active multiplication of HBV. The changes of the serological markers of HBV may be not consistent with that of HBV DNA. It is more objective to undergo both internal-standard and external-quantitative standard methods.

[A family with familial dysalbuminaemic hyperthyroxinaemia].

OBJECTIVE: To report a family of familial dysalbuminaemic hyperthyroxinaemia(FDH). METHODS: Four members, including the female proband, mother, daughter and brother, went through the measurement of thyroid hormone and thyroid-stimulating hormone (TSH). Electrophoretic analysis of the patient's serum proteins was carried out after the patient's serum being incubated with fluorescein isothiocyanate (FITC) labeled thyroxine(T4), The point mutation of Alb gene was determined in all members. RESULTS: The measurements of thyroid hormane and TSH showed that in three members (the proband, her mother and her daughter), the total thyroxine(TT4) serum level was high, the total triiodothyronine(TT3), FT4, FT3 and TSH serum levels were normal. And the enhanced albumin binding of fluorescenced T4 by electrophoresis showed a mutation transition 653 G-->A on DNA coding region of albumin. But in the proband's brother, the thyroid function and the results of electrophoresis of thyroxine-binding protein and determination of albumin gene were normal. CONCLUSION: A family with FDH in China is firstly reported here, a mutation at albumin gene DNA coding region 653G-->A causing enhanced albumin binding of T4 results in high T4 level.