Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. Objective: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. Methods: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained the advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance maintained high levels. Conclusion: Indinavir- resistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.

Objective To characterize 5 gpl20 sequences from mainly circulating clades in China and expression of their gp120 glycoproteins. Methods gp120 genes were amplified from the PBMCs of 5 HIV-1 infected individuals in different provinces using nest PCR and their DNA sequences were determined. Sequence characteristics were analyzed and gp120 genes were sub-cloned into the mammalian expression vec-tor to produce gp120 glycoproteins. Results Sequence characteristics indicated these sequences belong to the clade Thai-B, CRF_BC and CRF_AE, respectively. There were some conservative N-linked glycosyla-tion sites and primary Furin protease cleavage motifs in the same positions within gp120 amino acid se-quences although these gp120 sequences were categorized into different clades. In comparison with referen-tial strains, amino acids deletions were found in the V1, V2, V4, V5 regions except for the V3 loop; above all, V3 tip motifs of Thai-B exhibited the more polymorphic forms than those of CRF_BC and CRF_AE. These 5 gp120 sequences were cloned into the eukaryotic expression vector and gpl20 glycoproteins were produced successfully. Conclusion Hyper-variable nature of Env should be considered while designing HIV-1 vaccine or test reagent, and gpl20 expression in vitro is helpful to further research on the Env patho-genesis and vaccine development against the mainly circulating HIV-1 isolates in China.

ObjectiveTo evaluate the sensitivity and accuracy of an in-house detecting method of HIV-1 genotypic drug resistance system. MethodsTotally 130 serum specimens from Henan and Guangxi province were collected from April 2004 to October 2008 and tested in the Military HIV Testing Center of China. ViroSeqTM v2.0 (Abbott, Switzerland), a US FDA approved HIV genotypic drug resistance detecting system was utilized as the reference method. All the specimens were detected by the novel in-house method and the reference method to validate the difference in amplifying efficiency, drug resistance mutation detection and drug resistance report. ResultsConcerning the 14 850 known drug resistance mutation sites,14 752 (99. 3％ ) mutations can be detected by both of the two methods. Rates of concordance of detection in the regions of protease inhibitors-, reverse transcriptase inhibitors- and both two classes inhibitors-resistance were99.7％ ( Kappa =0. 909 9 , P ＜0. 01 ) , 99. 0％ (Kappa=0.952 1, P＜0. 01) and99.3％ (Kappa=0. 948 8, P ＜ 0. 01 ) respectively. Drug resistance reports from these two systems showed similar results (Kappa = 0. 637 4, P ＜ 0. 01 ). The in-house detecting system identified 34 novel mutations besides the ViroSeqTM drug resistance mutation database ( ViroSeqTM software v2. 7). Two mutations, V179F and K238T,had significant effect on HIV drug resistance. ConclusionsThe in-house genotyping system is an accurate,cost-effective method and has a high concordance with commercial ViroSeqTM genotyping system. Database from the in-house assay was superior to this of the ViroSeqTM assay.

Objective To develop and evaluate the allele-specific real-time PCR(ASPCR) assay for the detection of minor HIV-1 variants. Methods We developed and evaluated the ASPCR assay, using the K103N mutation site as a model system. We constructed plasmids as standards and designed specific and non-specific primers to discriminate the wild-type and mutant plasmids in the real-time PCR using SYBR green as fluorescence reporter. And then we evaluated the sensitivity, accuracy, reproducibility of ASPCR assay and detected the control samples. Results The specific primer can discriminate the wild-type and mutant plasmids including resistant mutation successfully. The sensitivity of ASPCR assay can achieve less than 0.01% and the accuracy of this method is down to 0.1%. The Intra-assay coefficient of variation is less than 0.7 and the Inter-assay coefficient of variation is less than 1.6. Conclusion ASPCR is a sensitive, accurate and rapid method to detect the minor HIV-1 variants which have resistant mutations and it can be used widely in HIV research. ASPCR also can provide earlier and more resistant information to the clinical therapy.

Objective To evaluate the antiretroviral therapy(ART),analyze the prevalence of resistance in rural areas,Henan,and explore the presence of minor resistant variants in pre-ART.Methods One hundred and forty-nine AIDS patients initiating ART were recruited and investigated at intervals of 6 months. Method of In-house developed by our laboratory for genotypjc resistance test was to analyze the occurrence of resistance among the failure of ART,and the allele-specific real.time PCR(ASPCR)was used to detect the minor resistant variants at the baseline samples once the resistance occurred.Results Vimlload significantly decreased among the patients who received ART(t=275,P=0.0001),but the absolute counts of CD4+T lymphocytes had no significant change(t=1.765 168,P=0.0852).Rate of resistance among the patients of treatment failure was 4.88%.The result of ASPCR in the survey of baseline showed that the minor resistant variants of M184V were detected in 7 patients and mutation K103N presented in 5 patients.Conclusion The primary drug-resistant straias in the untreated patients were found in Henan,and they might develop the dominant resistance strains and bring about the failure of ART.

Objective To elucidate the molecular evolutional characteristics of HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance-associated mutations in AIDS patients receiving highly active antiretroviral therapy (HAART).Methods Four AIDS patients receiving HAART with good adherence within a HlV-1 drug resistance cohort from a rural region in central China were selected,who possessed susceptible virus at the beginning of treatment and gradually came to produce resistance to NNRTIs during the process of antiretroviral therapy (ART),reverse transcriptase (RT) genes from each patient's peripheral blood samples (from 3 to 30 months after withdrawal) were cloned and sequenced in succession.Results To sequenced total 855 clones and obtained the HIV-1 NNRTI drug resistance-asseciated mutations patterns of the four patients: (1)G190A often appeared with F227 L and had the tendency of accumulating P236V during the process of treatmenL (2)Y188C always presented alone and sometimes it concured with P236V.(3) YI81C frequently concured with VI79D or KIO3N and the combination varies from patient to patient.(4)K103N often combined with Y181C or M230L Conclusions The molecular evolutional characteristics of HIV-1 NNRTI drug resistance-asseciated mutations in the 4 AIDS patients are summarized.They showed different pathways on HIV-1 NNRTI drug resistance-associated mutations and those mutations detected early tend to be predominant strains.

Neutralizing antibodies are considered to be an important protective parameter used in HIV-l vaccine evaluation.However,the exact role that neutralizing antibodies plays in controlling the disease progression of HIV-1 infected peoples is still undetermined.In this paper,we compared the protective function of the neutralizing antibody response in the plasma from LTNP and TP against clade B and clade C pseudoviruses.No difference in the neutralizing activities between the plasma from LTNP and TP was found,which was consistent with the most recent reports.In addition,no correlations between the titer or breadth and CD4+ or viral load in HIV-1 infected individuals were found.The protective roles played by neutralizing antibodies in controlling disease progression of HIV-1 infected people need to be considered in a new viewpoint.

Objective To isolate stable passage primary HIV-1 drug resistant strains and observe replication dynamics of the drug resistant isolates and evolvement tendency of the drug resistant mutations in vitro.Methods Peripheral blood mononuclear cells(PBMCs)from 15 AIDS patients receiving highly active antiretroviral therapy(HAART)were collected,and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people.HIV-1 pol genes from those strains were obtained by RT-PCR and sequenced.The drug resistant mutations were analyzed in the Stanford HIV Drug Resistance Database.Results Eight strong positive strains were isolated from 15 AIDS patients with viral loads higher than 1000 copies/ml,and two of them were drug resistant.Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP,respectively.K103N,M184V,Y181C and H221Y exist stably in the environment without drug pressure,however,RT K238T reverted to K238.Conclusion Two drug resistant strains were successfully isolated in vitro without drug pressure.Strains with K103N shows superior fitness and can exist steadily.Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure.NNRTI mutation K238T reproduces astatically,which suggests that RT 238 codon might revert gradually to wild genotype.

Vestibular schwannomas (VSs) are the most common cerebellopontine tumors. The natural history of smaller-sized VSs (<30 mm) has been well-studied, leading to the recommendation of a “watch and wait” approach. However, large VSs (>30 mm) have not been extensively studied, mainly because of their rarity. As such, most patients are conventionally offered surgery which carries a significant risk of neurological morbidity. Here, we report a case of a giant VS (>40 mm) in a 30-year-old man who regressed spontaneously. He was lost to follow-up for 18 years and, upon re-presentation, the symptomatology drastically improved and repeat imaging demonstrated a marked reduction in tumor size. Referring to similar cases in other studies, we postulate that most large and giant VSs undergo a phase of growth and stasis, followed by regression due to shifts in the balance between tumorigenic and regressive factors. Taken together with emerging molecular data, further studies are required to better understand the history of large and giant VSs to shape more personalized treatment options. This potentially includes non-operative management as a tenable option.

OBJECTIVE: The large sample retrospective cohort study were used to compare the diagnostic efficiency of PET/CT with conventional work-up (CWU) for evaluating nasopharyngeal carcinoma (NPC) distant metastasis. METHOD: Five hundred and fourteen patients with NPC were divided into PET/CT group and CWU group according the method of detecting distant metastasis. Chest film, abdominal ultrasonography, and bone scan were used in CWU group. Then the diagnostic efficiency of the two groups was compared. RESULT: Two hundred and sixteen patints were enrolled in PET/CT group and two hundred and nineteen-eight ones in CWU group. There were 28 out of 412 suspicious patients in CWU group were confirmed, another 3 patients confirmed without positive findings, compared with PET/CT group that all 32 suspicious patients were confirmed. The sensitivity and specificity of PET/CT were 100.0% (32/32) and 100.0% (184/184), as compared to 90.3% (28/31) and 94.8% (253/267) with CWU respectively, while there was no statistical significance. Further research found out that the percentage of patients with multiple distant metastatic sites and multiple organ metastases was higher in PET/CT group (P < 0.05), and similarly of patients with distant metastasis in N2-3 stages (P < 0.01). CONCLUSION Our results suggest that PET/CT appears to be slightly superior to conventional work-up in assessment of distant metastasis in NPC patients, but CWU is still a cheap and practical method.
Aged ; Carcinoma ; Cohort Studies ; Female ; Fluorodeoxyglucose F18 ; Humans ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; diagnosis ; secondary ; Positron-Emission Tomography ; methods ; Radiopharmaceuticals ; Retrospective Studies ; Sensitivity and Specificity ; Tomography, X-Ray Computed ; Ultrasonography