Objective: To understand the immunological and virological characteristics of HIV-1 infected men who have sex with men (MSM) in the acute phase in Tianjin and evaluate the effects of the fourth generation HIV ELISA and the P24 ELISA for acute HIV-1 infected samples. Methods: From October 2015 to October 2016, MSM were recruited through the community-based organizations in Tianjin. All the participants received rapid HIV test, positive samples were confirmed by Western Blot and negative samples underwent pooled nucleic acid testing. The participants with HIV-1 RNA reactive result underwent testing for viral load and T-cell count after second blood collection. Acute HIV-1 infection was defined as negative rapid HIV test result and the positive results of two HIV RNA tests, then the sensitivity were compared between the fourth generation HIV ELISA and the P24 ELISA to detect the initial HIV-1 RNA positive samples. Results: Among 3 016 MSM screened, 193 were positive in rapid HIV test. Western blot testing indicated that 179 cases were HIV positive, 7 cases were HIV indeterminate and 7 cases were negative. Of 2 823 sero-negative cases, 17 were acute HIV-1 infections. The HIV-1 infection rate was 6.53% (197/3 016) and the acute HIV-1 infection rate was 0.56% (17/3 016), with an average viral load of (5.63±1.50) log(10) copies/ml, an average CD(4) count of (442.82±268.17) cells/μl, an average CD(8) count of (1 069.65±668.22) cells/μl and an average CD(4)/CD(8) ratio of (0.49±0.25). Higher viral load, CD(4) and CD(4)/CD(8) ratio were seen in the acute HIV-1 infection group compared with the chronic HIV-1 infection group (U=148, P<0.01; U=272, P=0.042 and t=3.147, P=0.005). Demographic characteristics were similar between two groups, except the occupation (χ(2)=11.016, P=0.026). The sensitivity of P24 ELISA was higher than the fourth generation HIV ELISA in the HIV-1 detection for acute infection (Fisher's exact test, P=0.017). Conclusions: MSM are at risk for acute HIV-1 infection. Screening for acute HIV-1 infection with P24 ELISA would increase the sensitivity of diagnosis and reduce HIV transmission in MSM.
Adult ; Asian People ; HIV Infections/epidemiology* ; HIV-1 ; Homosexuality, Male ; Humans ; Male ; Mass Screening/methods* ; Nucleic Acid Amplification Techniques ; Viral Load

Human cytomegalovirus (CMV) is a clinically important pathogen that causes significant morbidity and mortality in immunocompromised patients and is typically monitored using real-time polymerase chain reaction (real-time PCR). International standards and certified reference materials were recently developed by the WHO, providing the opportunity to standardize viral load reporting. Clinical microbiologists who conduct quantitative CMV DNA testing should be aware of technical issues that can affect the analytical and clinical performance of the method used. These include specimen type, limits of detection and quantification, linear range, reproducibility, and wide variability in viral load values among different assays. Specimen types for testing include whole blood, plasma, serum, and peripheral blood leukocytes. The tests that use whole blood and peripheral blood leukocytes have higher sensitivities. Adsorption chromatography methods are widely used for nucleic acid extraction, and efficiencies can differ between manual and automatic processes. The most common method for quantitative CMV DNA testing is real-time PCR. All CMV testing methods require quality control at the pre-analytic, analytic, and post-analytic stages. In transplant patients, specific quantitative results and monitoring of any changes at follow-up are important. Five to seven days is an adequate follow-up interval in this regard, and drug-resistant CMV should be suspected if there is no response to therapy. One specimen type should be chosen for follow-up quantitative CMV DNA testing, optimized according to WHO standards. Further studies are needed to better standardize CMV testing approaches and to determine the appropriate clinical cut-off level.
Adsorption ; Chromatography ; Cytomegalovirus* ; DNA* ; Follow-Up Studies ; Humans ; Immunocompromised Host ; Leukocytes ; Limit of Detection ; Methods ; Mortality ; Plasma ; Polymerase Chain Reaction* ; Quality Control ; Real-Time Polymerase Chain Reaction ; Viral Load

OBJECTIVE: This study was conducted using the human papillomavirus (HPV) DNA chip test (HDC), in order to determine whether the HPV genotype is a predictor of residual disease in a subsequent hysterectomy following a loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia (CIN) 3. METHODS: Between January 2002 and February 2015, a total of 189 patients who underwent a hysterectomy within 6 months of LEEP caused by CIN 3 were included in this study. We analyzed their epidemiological data, pathological parameters, high-risk HPV (HR-HPV) load as measured by the hybrid capture II assay, and HR-HPV genotype as measured by the HDC. A logistic regression model was used to analyze the relationship between covariates and the probability of residual disease in subsequent hysterectomy specimens. RESULTS: Of the 189 patients, 92 (48.7%) had residual disease in the hysterectomy specimen, CIN 2 in seven patients, CIN 3 in 79 patients, IA1 cancer in five patients, and IA2 cancer in one patient. Using multivariate analysis, the results were as follows: cone margin positivity (odds ratio [OR], 2.43; 95% CI, 1.18 to 5.29; p<0.05), HPV viral load > or =220 relative light unit (OR, 2.98; 95% CI, 1.38 to 6.43; p<0.01), positive endocervical cytology (OR, 8.97; 95% CI, 3.81 to 21.13; p<0.001), and HPV-16 or HPV-18 positivity (OR, 9.07; 95% CI, 3.86 to 21.30; p<0.001). CONCLUSION: The HPV-16 or HPV-18 genotype is a reliable predictive factor of residual disease in a subsequent hysterectomy following a LEEP for CIN 3.
Adult ; Aged ; Aged, 80 and over ; Cervical Intraepithelial Neoplasia/*surgery/virology ; Electrosurgery/methods ; Female ; Genotype ; Genotyping Techniques/methods ; Human papillomavirus 16/genetics/*isolation & purification ; Human papillomavirus 18/genetics/*isolation & purification ; Humans ; Hysterectomy ; Middle Aged ; Neoplasm, Residual ; Papillomavirus Infections/*virology ; Prognosis ; Retrospective Studies ; Uterine Cervical Neoplasms/*surgery/virology ; Viral Load

OBJECTIVE: The aim of this study was to describe the principle of the Cheil HPV DNA Chip assay and evaluate its accuracy. In order to quantify the human papillomavirus (HPV) load and identify HPV genotypes simultaneously, this assay combined the two methods: SYBR Green quantitative real-time polymerase chain reaction (PCR) and DNA microarray. METHODS: We designed novel consensus primer sets that target the conserved region of the HPV L1 gene for quantifying and detecting a broad range of HPV types by quantitative real-time PCR. Subsequently, using the PCR products, DNA microarray was performed with 36 HPV type-specific probes. To validate this method, direct sequencing and correlation analysis among HPV genotype, viral load, and cytological abnormality was performed by Cohen’s kappa values, two-sided McNemar chi-square test, Kruskal-Wallis test, and odds ratios. RESULTS: The kappa value of the Cheil HPV DNA Chip was 0.963 (95% confidence interval, 0.919 to 0.98), which was significantly higher than the value of 0.527 (95% confidence interval, 0.447 to 0.59) obtained using a conventional HPV DNA Chip. HPV16 (χ²=62.28, P<0.01), HPV33 (χ²=7.18, P<0.01), and HPV58 (χ²=9.52, P<0.01), which are classified as high-risk HPVs, were detected at significant levels in samples with high-grade lesions. And viral loads tended to be higher in groups with high odds ratios. CONCLUSION: The Cheil HPV DNA Chip is an effective diagnostic assay for simultaneously detecting HPV genotypes and loads in cervical samples.
Consensus ; Diagnosis ; Genotype* ; Humans* ; Methods* ; Odds Ratio ; Oligonucleotide Array Sequence Analysis ; Papillomaviridae ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction* ; Viral Load

BACKGROUNDRheumatic diseases involve multiple organs that are affected by immunological mechanisms. Treatment with corticosteroids and immunosuppressive agents may also increase the frequency of infection. Cytomegalovirus (CMV) is a widespread herpes virus and a well-recognized pathogen, which causes an opportunistic and potentially fatal infection in immunocompromised patients. This retrospective study aimed to investigate the clinical and laboratory characteristics of CMV pneumonia in patients with rheumatic diseases after immunosuppressive therapy in a single center in Shanghai, China.

METHODSEight hundred and thirty-four patients with rheumatic diseases who had undergone CMV-DNA viral load tests were included, and the medical records of 142 patients who were positive for CMV-DNA in plasma samples were evaluated. GraphPad Prism version 5.013 (San Diego, CA, USA) was used to conduct statistical analysis. The correlation between CMV-DNA viral loads and lymphocyte counts was assessed using the Spearman rank correlation coefficient test. Significance between qualitative data was analyzed using Pearson's Chi-squared test. The cut-off thresholds for CMV-DNA viral load and lymphocyte count were determined by receiver operating characteristic (ROC) curve analysis.

RESULTSOne hundred and forty-two patients had positive CMV viral load tests. Of these 142 patients, 73 patients with CMV pneumonia were regarded as symptomatic, and the other 69 were asymptomatic. The symptomatic group received higher doses of prednisolone (PSL) and more frequently immunosuppressants than the asymptomatic group (P < 0.01). The symptomatic group had lower lymphocyte counts, especially CD4+ T-cells, than the asymptomatic group (P < 0.01). By ROC curve analysis, when CD4+ T-cell count was <0.39 × 109/L, patients with rheumatic diseases were at high risk for symptomatic CMV infection. The CMV-DNA load was significantly higher in the symptomatic patients than that in asymptomatic patients (P < 0.01; threshold viral loads: 1.75 × 104 copies/ml). Seven patients had a fatal outcome, and they had lower peripheral lymphocyte counts (P < 0.01), including CD4+ and CD8+ T-cells (P < 0.01).

CONCLUSIONSWhen CD4+ T-cell count is <0.39 × 109/L, patients are at high risk for pulmonary CMV infection. Patients are prone to be symptomatic with CMV-DNA load >1.75 × 104 copies/ml. Lymphopenia (especially CD4+ T-cells), presence of symptoms, and other infections, especially fungal infection, are significant risk factors for poor outcome, and a higher PSL dosage combined with immunosuppressants may predict CMV pneumonia.

CD4-Positive T-Lymphocytes ; metabolism ; China ; Cytomegalovirus ; pathogenicity ; Cytomegalovirus Infections ; genetics ; immunology ; therapy ; virology ; Humans ; Immunosuppression ; methods ; Pneumonia ; genetics ; immunology ; therapy ; virology ; Polymerase Chain Reaction ; Retrospective Studies ; Rheumatic Diseases ; genetics ; immunology ; therapy ; virology ; Viral Load

BACKGROUND/AIMS: Advanced human immunodeficiency virus (HIV) infection, despite sustained viral suppression by highly active antiretroviral therapy (HAART), is a risk factor for poor immunologic recovery. However, some patients with advanced infection do show immunologic recovery. In this study, predictive factors of immunologic recovery were analyzed in advanced HIV patients showing sustained viral suppression. METHODS: A case-control study was conducted in HIV-infected adult patients with HIV-1 RNA < 50 copies/mL maintained for 4 years or longer and who were receiving HAART. Advanced HIV infection was defined as a baseline CD4 T cell count < 200/mm3. Immunologic responders were defined as patients showing immunologic recovery (CD4 T cell counts > or = 500/mm3 at 4 years with HAART). To analyze the CD4 T cell kinetics, the CD4 slope (monthly changes in the CD4 T cell count) was estimated for each patient using a linear regression between the CD4 T cell count and the time since HAART initiation. RESULTS: Of 102 eligible patients, 73 had advanced HIV, and 33 (45.2%) showed immunologic recovery. The median CD4 slopes (cells/mm3 per month) during 0 to 6 and 0 to 12 months of HAART in the 73 advanced patients were significantly higher in responders than in non-responders (0 to 6 months, 38.6 vs. 22.8; 0 to 12 months, 24.5 vs. 13.5). Multivariate analyses showed opportunistic infections at the start of HAART (adjusted odds ratio [OR], 0.28) and a CD4 slope > or = 20 during 0 to 12 months of HAART (adjusted OR, 10.10) were independently associated with immunologic recovery. CONCLUSIONS: The CD4 slope can be an early predictor of long-term immunologic recovery in advanced HIV patients.
Adult ; Anti-HIV Agents/therapeutic use ; Antiretroviral Therapy, Highly Active ; Biomarkers/blood ; *CD4 Lymphocyte Count ; Case-Control Studies ; Chi-Square Distribution ; Female ; HIV Infections/*diagnosis/drug therapy/*immunology/virology ; HIV-1/drug effects/genetics/*immunology ; Humans ; Linear Models ; Logistic Models ; Male ; Middle Aged ; Monitoring, Immunologic/*methods ; Multivariate Analysis ; Odds Ratio ; Predictive Value of Tests ; RNA, Viral/blood ; Time Factors ; Treatment Outcome ; Viral Load

PURPOSE: Landmark indicators have not yet to be developed to detect the regression of cervical intraepithelial neoplasia (CIN). We propose that quantitative viral load and indicative histological criteria can be used to differentiate between atypical squamous cells of undetermined significance (ASCUS) and a CIN of grade 1. MATERIALS AND METHODS: We collected 115 tissue biopsies from women who tested positive for the human papilloma virus (HPV). Nine morphological parameters including nuclear size, perinuclear halo, hyperchromasia, typical koilocyte (TK), abortive koilocyte (AK), bi-/multi-nucleation, keratohyaline granules, inflammation, and dyskeratosis were examined for each case. Correlation analyses, cumulative logistic regression, and binary logistic regression were used to determine optimal cut-off values of HPV copy numbers. The parameters TK, perinuclear halo, multi-nucleation, and nuclear size were significantly correlated quantitatively to HPV copy number. RESULTS: An HPV loading number of 58.9 and AK number of 20 were optimal to discriminate between negative and subtle findings in biopsies. An HPV loading number of 271.49 and AK of 20 were optimal for discriminating between equivocal changes and obvious koilocytosis. CONCLUSION: We propose that a squamous epithelial lesion with AK of >20 and quantitative HPV copy number between 58.9-271.49 represents a new spectrum of subtle pathological findings, characterized by AK in ASCUS. This can be described as a distinct entity and called "regressing koilocytosis".
Biopsy* ; Cervical Intraepithelial Neoplasia ; Female ; Humans* ; Inflammation ; Logistic Models ; Methods ; Papilloma* ; Viral Load ; Viruses*

OBJECTIVETo evaluate the value of high risk (HR)-HPV viral load in predicting cervical lesions and triaging for HR-HPV positive women.

METHODSThe study cohort came from a multicenter cervical cancer screening program. HR-HPV was detected by hybrid capture 2 (HC-2) assay, and viral load was measured by the ratio of relative light units to cut off (RLU/CO). Women were divided into 4 groups according to the RLU/CO value, and CIN diagnostic system was used to describe the severity of cervical lesions. Chi-square trend test was used to analyze the association between viral load and CIN. The absolute and relative risks of CIN2+ in different viral load groups were calculated, and the clinical performance to detect CIN2+ at follow-up by different cut-off values of baseline RLU/CO was also calculated.

RESULTS2 725 women with complete results of both baseline and follow-up were included in this analysis. The severity of cervical lesions increased with the increasing viral load (P < 0.001). In women with normal or CIN1 diagnosis at baseline, the absolute risk of one-year accumulative CIN2+ was 0.11% in the HR-HPV-negative group, compared with 3.14% in the moderate viral load group and 6.09% in the high viral load group, and the relative risk of 29.05 (95%CI: 6.07-138.99) in the moderate viral load group and 56.34 (95%CI: 12.89-246.30) in the high viral load group. Raising cut-off value of baseline HR-HPV viral load to 15.00, RLU/CO decreased the number of women who need to be followed up at one-year from 774 to 412, with the sensitivity of 91.30% and specificity of 47.94% in detecting CIN2+ at follow-up.

CONCLUSIONSThe risk of cervical cancer and precancerous lesions increases with the increasing HR-HPV viral load. Raising the cut-off value of HR-HPV viral load can triage for HR-HPV-positive women, therefore help to allocate the health resources more effectively.

Adult ; Aged ; Cervical Intraepithelial Neoplasia ; pathology ; virology ; Disease Progression ; Female ; Follow-Up Studies ; Humans ; Mass Screening ; methods ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; Risk Factors ; Uterine Cervical Neoplasms ; pathology ; virology ; Viral Load

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
Bunyaviridae Infections ; diagnosis ; virology ; Cell Line ; DNA Ligases ; metabolism ; DNA, Complementary ; analysis ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Humans ; Phlebovirus ; genetics ; isolation & purification ; RNA, Viral ; analysis ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Load

To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.
Adolescent ; Adult ; Aged ; Conserved Sequence ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; virology ; DNA, Viral ; blood ; Female ; Humans ; Immunosuppressive Agents ; blood ; Kidney Transplantation ; adverse effects ; Male ; Middle Aged ; Polyomavirus ; genetics ; isolation & purification ; Polyomavirus Infections ; diagnosis ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Species Specificity ; Tacrolimus ; blood ; Time Factors ; Tumor Virus Infections ; diagnosis ; virology ; Viral Load ; Young Adult