Recent clinical research on the application of liquid biopsy in neuroblastoma / 中国当代儿科杂志

Neuroblastoma (NB) is the most common extracranial solid tumor in children and has the features of high recurrence rate and low survival rate, and therefore, early diagnosis, treatment response evaluation, and recurrence monitoring are of great significance for NB patients. Liquid biopsy refers to the detection of cells and nucleic acids in fluid specimens, mainly blood. It is noninvasive and can overcome tumor heterogeneity, thus making it possible to achieve the early diagnosis and dynamic detection of NB. This review introduces the latest advances in clinical research on the application of liquid biopsy in NB.

Expression and effect of TLR4 in rats with diabetic nephropathy

OBJECTIVE@#To observe the expression of TLR4 in kidney tissue of rats with diabetic nephropathy and discuss the role of TLR4 in the occurrence and development of the diabetic nephropathy.@*METHODS@#A total of 60 clean male SD rats were selected and randomly divided into the modeling group and control group after 1 week of breeding, including 30 rats in each group. Biochemical indices as well as the protein expression of TLR4 were observed and compared between two groups at 2 w, 4 w, 6 w, 8 w and 12 w after the modeling, and the correlation between TLR4 and each biochemical indexes was analyzed.@*RESULTS@#Rats in the modeling group had higher levels of blood glucose, 24-hour urine protein and blood urea nitrogen after the modeling, and showed the increase in the serum creatinine, kidney/body weight ratio, CRP and serum TNF-α at 4w after the modeling, with the significant difference compared to results of the control group (P<0.05). The cross-section area and mean volume of glomerulus in the modeling group at 4 w, 6 w, 8 w and 12 w were significantly higher than those in the control group, with the statistically significant difference (P<0.05). The expression of TLR4 at each time point in the control group was relatively low. Rats in the modeling group had the high expression of TLR4 in kidney's glomerular basement membrane, proximal convoluted tubule and renal interstitial area since 2 w, with the significant difference compared to the control group (P<0.05). The expression in rats of the modeling group was higher than the one of the control group since the 2nd week. As the time flied, its expression increased, with the statistically significant difference between two groups (P<0.05). There was certain correlation between the protein expression of TLR4 and the increased serum titer of 24-hour urine protein excretion, serum creatinine, CRP and TNF-α.@*CONCLUSIONS@#TLR4 may activate the immuno-inflammatory reactions to play a role in the occurrence and development of the diabetic nephropathy.

Effect of rotation stimulation on the anesthetic sensitivity of sevoflurane in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effect of simulated navigation stimulation on the anesthetic sensitivity of sevoflurane in rats, so as to provide basis for rational using sevoflurane during navigation.</p><p><b>METHODS</b>SD rats were stimulated by Crampton model and the conditioned taste aversion (CTA) was regarded as criterion of motion sickness. (1) 60 rats were randomly divided into control (n = 15) and rotation group (n = 45). The changes of behavior and autonomic activity, sevoflurane concentration achieved sleep and anesthesia states, and the revitalization time were observed in two group rats. (2) 32 rats were randomly divided into control (I), rotation (II), anesthesia (III) and rotation plus anesthesia (IV) group (n = 8). The acetylcholine (Ach), norepinephrine (NE), r-aminobutyric acid (GABA), glutamic acid (Glu) of brain cortex, thalamus and hippocampus were determined respectively in the four group rats.</p><p><b>RESULTS</b>In control group, the sevoflurane concentration achieved sleep and anesthesia states were 1.74% +/- 0.05% and 3.54% +/- 0.05% respectively, but, those concentrations were 1.51% +/- 0.06% and 3.14% +/- 0.08% in rotation group. There were lower significantly in rotation group than those in control group (P < 0.01). It was a major characteristic that all of the neurotransmitters were reduced significantly in II group, this was even more in brain cortex and thalamus (P < 0.01). In II group, Ach was upward in brain cortex, NE and GABA were reduced in hippocampus obviously. The change tendency of neurotransmitters in IV group was more close to II group, that was, the effect of rotation stimulation was more obvious.</p><p><b>CONCLUSION</b>The anesthetic sensitivity of sevoflurane could be obvious increased in rats simulated navigation stimulation.</p>

Antiviral effect of lamivudine on HIV-1 targeting MT2 cells influenced by morphine / 中华流行病学杂志

Objective To determine whether morphine having the ability to influence the antiviral effect of lamivudine(3TC)in vitro study.Methods MT2 cells were randomly assigned into morphine+3TC treatment group,morphine+naloxone+3TC treatment group,naloxone+3TC treatment group.Both 3TC and virus control groups were set up.The corresponding MT2 cells were treated with opiates antagonist(naloxone)for 0.5 hours before the 24-hours morphine treatment program was implemented while all of the groups were then infected with equal amounts of cell-free HIV-1 ⅢB strain and 3TC.HIV-1 p24 antigen in culture supernatants collected at days 3,4,5 and 6after infection status was tested and the inhibition of 3TC anti-HIV-1 p24 antigen of various treatment groups calculated.Results Inhibition of 3TC anti-HIV-1 p24 antigen of Morphine+3TC treatment group was the lowest when HIV-1 infected cells at 3rd and 4th day and showed significant difierence (P<0.05)when compared to the 3TC control.However,there was no statistically significant difference among them(P>0.05),when virus was infected the cells at 5th and 6th day.The difference of 3TC anti-HIV-1 p24 antigen inhibition between the morphine+naloxone+3TC treatment group and the naloxone+3TC treatment group was not significant(P>0.05).Similar results were obtained when these two groups were compared to the 3TC control group(P>0.05),respectively.The 3TC anti-HIV-1 p24 antigen inhibition of each treatment group reduced as the time of infection prolonged,showing a significant and time-course effbct.Conclusion The 3TC antiviral effect was reduced by morphine in the early stage of infection,and could be blocked by naloxone.

Screening program on novel drug resistance mutations of subtype B' in human immunodeficiency virus type 1 in China / 中华流行病学杂志

Objective To screen the level of novel drug resistance mutations in subtype B' in China. Methods 451 pol sequences collected from the previous study, which including 354 AIDS patients who had received antiretroviral treatment(ART)and 97 the untreated patients. Entire protease gene(codous 1-99)and full-length reverse transcriptase gene(codons 1-560)were included.Variation of mutations between the treated and the untreated patients with consensus/ancestral sequences were compared and the mutations with higher frequencies in the treated patients than in the untreated patients were screened before submitting the mutations to the Stanford HIV Drug Resistance Database(SHDB)(http://hivdb.stanford.edu/). Relation between the mutations and resistance preliminarily was then analyzed, according to the information including SHDB. Results Frequencies of 7 mutations at 6 positions, DI23E, V292I, K366R, T369A, T369V, A371V and 1375V, 2 at DNA polymerase domain and 5 at connection domain of reverse transcriptase(RT)were higher in the treated patients than in the untreated patients. The information of 7 mutations including the SHDB showed that 7 mutations were major variants at corresponding positions, and theirs frequencies were higher in the treated patients using some drugs, than in the untreated patients. Conclusion 7mutations being screened from the China subtype B were possibly associated with the resistance,which called for the construction of mutated viruses by site-directed mutagenesis to identify their effects on the susceptivity of different drugs.

Studying on the prevalence and mutation pattern of N348I which related to the resistance of HIV-1 / 中华流行病学杂志

Objective To elucidate the prevalence and the mutation pattern of N348I that related to the resistance seen in the AIDS patients, in China. Methods Partial pol gene of HIV-1 comprising of full protease (PR) and reverse transeriptase (RT) was obtained from plasma samples of therapy-failure individuals (n=614) and therapy-naive individuals (n=619) by using reverse transcription polymerase chain reaction(RT-PCR). 1233 sequences were then submitted to the HIV-1 drug resistance database of the Stanford University to analyze the prevalence and the emergence pattern of N348I. Results The prevalence of N348I was 6.5％ in the therapy-failure patients and 0.8％ in the naive individuals, respectively. The prevalence of N348I was more popular among those patients whose ART regimens containing zidovudine (AZT or ZDV) than those without AZT in regimens( 14.1％ vs. 4.7％, x2=10.21, P＜0.01 ). N3481 always emerged, and combined with others mutations among patients of ART, whose frequencies were: 85.0％ in combination with thymidine analog mutations (TAMs) and 52.5％ with M184V/I, respectively. Conclusion N348I was somehow prevalent in the therapy-failure patients when using the first-line antiretroviral drugs,and it emerged as unique patterns. This study laid the ground in improving the techaology of drug resistance genotypes detection and providing theoretical basis to study the mechanism of resistance and the law of molecular evolution.

In vitro selection and identification of HIV strain which is resistance to two new HIV-1 nonnucleoside reverse transcriptase inhibitors / 药学学报

JB25 and JB26 are new HIV-1 nonnucleoside reverse transcriptase inhibitors, and show potent anti-HIV activities. Sequential passage experiments with wild-type virus were performed to select and identify mutations induced by these two compounds in vitro. For the initial passage, compounds were present at approximately 2-fold IC50 in MT-2 cells. When cytopathic effect (CPE) was observed in more than 75% of the cells, the culture supernatants were collected. For the subsequent passages, fresh MT-2 cells were infected with 1 mL supernatants from the previous passage (regardless of the virus titer) and cultured in the presence of the compounds at concentrations that were increased 2-fold compared with that in the previous passage. This procedure was repeated with increasing concentrations for 12 passages. JB25 had amino acid substitution L100I (TTA-->ATA) at passage 6, and then changed into 100 M (ATA-->ATG) at passage 12, which was rare mutation form and had not been reported. At the same time, Y188C (TAT-->TGT) mutation appeared at passage 10. For JB26, there was a L100I (TTA-->ATA) mutation at passage 10. In a word, JB25 and JB26 showed a low genetic barrier to the development of resistance, and the resistance to JB26 developed slower than JB25. The mutations selected by JB25 and JB26 were mainly associated with codons 188 and 100 of HIV-1 reverse transcriptase.

Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.</p><p><b>METHODS</b>We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method.</p><p><b>RESULTS</b>The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.</p><p><b>CONCLUSIONS</b>Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.</p>

Competitive capacity of HIV-1 strains carrying M184I or Y181I drug-resistant mutations / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Virus with nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs) resistant mutations show different evolution tendencies when the anti-viral therapies are interrupted. Understanding the replication fitness of drug-resistant virus is important for the study of the prevalence of drug-resistance. For this purpose, we characterized the replication capacity of HIV-1 virus carrying lamivudine (3TC) or nevirapine (NVP) resistant mutations.</p><p><b>METHODS</b>3TC and NVP resistant variants were induced in vitro by selecting wild type virus in the presence of drugs. For the competitive replication assay, drug-resistant variants were cocultured with wild-type virus in the presence or absence of drugs. The ratios of the viral species were determined over time by using a real-time RT-PCR-based assay.</p><p><b>RESULTS</b>3TC-resistant (M184I mutation) and NVP-resistant (Y181I mutation) virus should be selected in vitro in two different ways. The competitive replication assay showed that the ratio of virus carrying a M184I mutation increased from 98.8%, while the wild type virus decreased to 1.2% after 4 passages in the presence of 3TC; the percentage of virus carrying the Y181I mutation increased to 90.5%, while wild type virus decreased to 9.5% in the presence of NVP. In the absence of drugs, the ratio of virus carrying the M184I mutation decreased to 5.3%, while wild type virus increased to 94.7%; the ratio of virus carrying Y181I increased to 75%, while wild type virus decreased to 25% after 4 passages.</p><p><b>CONCLUSIONS</b>The NVP-resistant virus is fitter than wild type virus even in the absence of NVP that may be the reason that NNRTIs-resistant virus is spreading quickly.</p>

Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of "DNA prime plus protein boost" / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.</p><p><b>METHODS</b>Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.</p><p><b>RESULTS</b>Response of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.</p><p><b>CONCLUSION</b>Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.</p>

Anti-human immunodeficiency virus type 1 activity of two new nonnucleoside reverse transcriptase inhibitors in combination with other 3 antiretroviral agents in vitro / 军事医学科学院院刊

Objective:To evaluate the anti-HIV-1 activity of two new nonnucleoside reverse transcriptase inhibitors (NNRTIs), JB25 and JB26, in combination with 3 approved drugs (AZT, EFV, SQV)in vitro.Methods:The serially diluted 10 concentrations of JB25 and JB26 were combined with 7 serially diluted AZT, EFV and SQV respectively.The combination was added to 384 cell culture plates and then cocultured with HIV-1 ⅢB infected MT-2 cells for 3 days. Finally, the HIV-1 production was determined by measuring the expression of reporter genes of TZM bl cells. The data were analyzed by MacSynergy Ⅱ software.Results:The average capacity of synergism/antagonism of JB25 with AZT, EFV and SQV was 244.45/-5.05(nmol/L)~2%, 119.58/-65.93 (nmol/L)~2% and 145.83/-0.32 (nmol/L)~2% respectively;the average capacity of synergism/antagonism of JB26 with AZT, EFV and SQV was 398.90/0(nmol/L)~2%, 103.62/-0.49(nmol/L)~2% and 138.473/-0.27 (nmol/L)~2% respectively. Conclusion:Two new NNRTIs JB25 and JB26 develop synergism when combined with 3 approved drugs, respectively. MacSynergy Ⅱ software could evaluate the anti-HIV-1 activity of drug combination.

Subtype and characterization of gag gene of HIV-1 strains prevalent in some areas of China / 军事医学科学院院刊

Objective:To investigate the subtype distribution of HIV-1 strains prevalent in four areas of China,and to study the characteristics of gag gene variation and changes in antigen epitopes under the host immune pressures. Methods:The plasma of HIV-1 infected people from Henan, Guangdong, Sichuan and Beijing in China were collected. Virion RNA was extracted directly from plasma after the virion was condensed. The gag gene was amplified by RT-PCR and nested-PCR.Sequences were subtyped by Genotyping Tool software, and phylogenetic analysis of gag gene were performed using the MEGA 4.1 software.The gene distances intra each subtype were calculated by Distance program. The Ks/Ka ratios were calculated using SNAP program. The variation analysis of CTL antigen epitopes restricted by main HLA-Ⅰ specificities in China was performed.Results:Six subtypes or circulating recombinant forms(CRFs)of HIV-1,including B',CRF07_BC,CRF01_AE,B,CRF08_BC and CRF02_AG,were identified in four areas of China.The gene distances intra each subtype were CRF01_AE>B>CRF08_BC> CRF07_BC>B' listed in order of size, meanwhile the order of Ks/Ka ratios was CRF01_AE>B>CRF08_BC>B'>CRF07_BC. Far more diversity of antigen epitopes in P17 region was observed than that in P24.Epitope mutations intra subtypes were CRF01_AE>B>B'>CRF07_BC listed in order of size. Conclusion:Itseems that CRF01_AE is under the strongest immune pressures,and displays the most diversity of gene and variation of epitopes intra subtypes prevalent in China, followed by subtype B, B' and CRF07_BC. The discrepancy of epitope mutations intra the subtypes is significant.

Research on the selective kinetics of HIV-1 nucleoside reverse transcriptase inhibitor drug resistance-associated mutations among 4 AIDS patients receiving highly active antiretroviral therapy / 中华流行病学杂志

<p><b>OBJECTIVE</b>To elucidate the molecular evolutional characteristics of HIV-1 nucleoside reverse transcriptase inhibitor (NRTI) drug resistance-associated mutations in patients with AIDS receiving highly active antiretroviral therapy.</p><p><b>METHODS</b>We selected 4 AIDS patients receiving highly active antiretroviral therapy (HAART) with good adherence under a HIV-1 drug resistance cohort from a rural region in central China. Those people carried susceptible virus at the beginning of treatment and gradually came to produce virus resistant to NRTIs during the process of antiretroviral therapy (ART). Reverse transcriptase (RT) genes from each patient's peripheral blood samples (from 3 to 33 months after withdrawal) were cloned and sequenced in succession.</p><p><b>RESULTS</b>We sequenced a total number of 855 clones and obtained the HIV-1 NRTI drug resistance-associated mutations patterns of the 4 patients. Typical resistance mutations of thymidine analogue mutations (TAMs) pattern 1, such as L210W, T215Y and M41L, were generated in patient 'A'. TAMs pattern 2, including D67N, K70R and K219Q mutations, was discovered in patient 'B'. Interestingly, in patient 'C', some clones comprising not only TAMs pattern 1 mutations (T215Y) but also TAMs pattern 2 mutations (K70R, D67N).</p><p><b>CONCLUSION</b>The four patients show different pathways on HIV-1 NRTI drug resistance-associated mutations, including TAMs pattern 1, TAMs pattern 2 and the fusion pattern of TAMs-1 & TAMs-2. We also noticed that the tendency of gradual accumulation was obvious and those mutations detected earlier tended to be the predominant strains.</p>

Study on the difference of the APOBEC3G mRNA levels among HIV long-term nonprogressors, slow progressors, people at high risks and normal people / 中华流行病学杂志

Objective To examine the APOBEC3G(hA3G)mRNA levels of four different groups in the human immunodeficiency virus(HIV)prevalent areas in central China and to analyze the relationship between hA3G mRNA levels and HIV disease progression.Methods We collected peripheral blood and isolated the peripheral boold monouuclear cells(PBMCs),and then cryo-preserved the PBMCs in liquid nitrogen.Prior to the total extraction of RNA,PBMCs were resuscitated and mRNA were reverse Transcripted to cDNA in vitro.Real-time polymerase chain reaction(PCR)was used to test hA3G mRNA levels of different groups.Results There were 13 HIV long term non-progressors with the mean CD+4 T lymphocyte count as(716±169)per μl and the mean affection time as(12.5±2.3)years.There were 48HIV slow progressors with the mean CD+4 T lymphocyte count as(233±144)per μl and the mean affection time as(10.7±2.2)years.The hA3G mRNA level of HIV long term nonprogressors was higher than that of normal people while the hA3G mRNA level of HIV slow progressors was higher than that of normal people and high risk people.There were no correlations between CD+4 T lymphocyte count and hA3G mRNA levels of HIV long term nonprogressors as well as in HIV slow progressors.Conclusion There was difference found in the hA3G mRNA levels of four groups in the HIV popular area in central China while no correlation between CD+4 T lymphocyte count and hA3G mRNA levels of HIV long term nonprogressors as well as in HIV slow progressors were found.

Study on the antiviral therapy program among people with human immunodeficiency virus in Guangxi Zhuang Authority / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the reconstitution of immune function and viral suppression condition and to analyze the occurrence of drug resistance HIV-1 variants and its prevalence after using HAART in Guangxi Autonomy Region.</p><p><b>METHODS</b>From July 2004 to October 2005, 133 HIV infected individuals who had received HAART for more than three months were recruited, and 58 infected persons with no antiviral therapy were selected as controls. Questionnaire was used to collect information about the adherence of HAART therapy. Immune reconstruction and viral suppress conditions were obtained by CD4+ counts and viral load and RT-PCR were used to amplify the PR and RT regions of HIV-1 genome while HIV-1 drug resistance rates were analyzed to show the occurrence and prevalence in both treated and naive patients.</p><p><b>RESULTS</b>In terms of CD4+ T cell counts: 70.69% of the treated patients showed obvious increase and 23.28% had no apparent change but 6.03% of them went down. 70.48% of the patients who had received antiviral therapy more than 3 months had their viral load lower than the low detectable limitation. When comparing the log of viral load between treated and untreated cohort, the mean value of the treated was obviously less than the untreated (P < 0.05). However,the result of drug resistance showed no obvious difference between the treated and untreated groups.</p><p><b>CONCLUSION</b>The antiviral therapy being used in Guangxi region, had achieved obvious effect on the reconstruction of immune system and the suppression of viral replication in vivo under good adherence while the occurrence of drug-resistant HIV strain did not show obvious difference between treated and naive patient groups.</p>

Phenotypic resistance of resistant strains of HIV type-1 subtype B in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>This study was aim to explore the characteristics of phenotypic resistance of resistant strains of HIV type-1 (HIV-1) subtype B and to compare the concordance between the phenotypic resistance and genotypic resistance.</p><p><b>METHODS</b>The genotypic resistance assay for the HIV-1 clinical isolates was performed. One isolate without resistance mutation was chosen as a drug-sensitive reference strain and seven subtype B isolates with resistance mutations were phenotypically tested. Fifty percent inhibitory concentrations (IC50) between resistant and sensitive viruses were compared. The resistance extent was determined by the folds of the increased IC50. The concordance between the phenotypic resistance and genotypic resistance was also analyzed.</p><p><b>RESULTS</b>IC50 of resistant isolates were 0.0006 - 0.1300 micromol/L for zidovudine (AZT), 0.0016 - 0.0390 micromol/L for lamivudine (3TC), 0.0104 - 0.4234 micromol/L for nevirapine (NVP), and 0.0163 - 0.1142 micromol/L for indinavir (IDV), respectively. Genotypic and phenotypic resistance assays indicated that the resistant strains were intermediately and highly resistant to nucleotide analog reverse transcriptase inhibitors and non-nucleotide analog reverse transcriptase inhibitors. The phenotypic assay was consistent with the genotypic assay. For measuring the potential resistance, the genotypic assay was more sensitive than the phenotypic. In evaluating the resistance to protease inhibitors, these two assays were discrepant.</p><p><b>CONCLUSIONS</b>Both the phenotypic and genotypic assays indicate that the resistant viruses exist in HIV-infected patients in China who have received treatment. Phenotypic and genotypic assays have high concordance, and the genotypic assay could replace the phenotypic assay to predict the HIV-1 resistance.</p>

Cohort study on human immunodeficiency virus discordant couples in the countryside of central China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a cohort of human immunodeficiency virus (HIV) discordant couples for follow-up studies and to collect data on frequency of HIV heterosexual transmission and related factors.</p><p><b>METHODS</b>A total of 52 HIV discordant couples were identified by face to face interview and serological testing, in which the HIV negative individuals had no HIV infection behaviors including injecting drug use, blood transfusion or having sexual partners other than his/her own wife/husband. Three times of follows-up studies were carried out in 0.5 year, 1 year and 2.5 years to collect information on their sexual practices and condom use through face to face interview together with 20 ml whole blood collected to test HIV antibody, CD4+ T cell count and viral load.</p><p><b>RESULTS</b>(1) In the period of 2.5 years follow-up, no HIV seroconversion and HIV transmission was found. (2) The frequencies of sexual intercourse between once per month to once per week were 65.4%, 72.9%, 71.7% and 80.0% at the time of cohort setup: 0.5 year, 1 year and 2.5 years of follow-up respectively. The rates of "occasional use" to "never use" condoms were 76.9%, 66.6%, 69.1% and 60.0% at the time of cohort setup as: 0.5 year, 1 year and 2.5 years of follow-up, respectively. No significant difference between different times of follow-up for sexual intercourse or condom use. (3) 85.4%, 66.6% and 60.0% of the HIV positive individuals kept their CD4+ T cell count stabilized or raised during the 0.5 year, 1 year and 2.5 years follow-up period, respectively. However, 66.7% of them showed stable or declined viral load in the period of 2.5 years follow-up. It appeared that stable or raised CD4+ T cell and the stable/declined viral load happened simultaneously.</p><p><b>CONCLUSION</b>No transmission was identified in this study. The stabilized CD4+ T cell count and viral load might be account for the reason of no transmission while the biological factors from host and virus related with transmission need to be further studied.</p>

Cloning and characterization of a full-length HIV-1 genome of a prevalent subtype B-Thai strain in Henan Province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software.</p><p><b>RESULTS</b>HIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42.</p><p><b>CONCLUSION</b>The cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.</p>

Study on factors associated with heterosexual-transmission of human immunodeficiency virus in central China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the factors of the human immunodeficiency virus (HIV) associated with sexual-transmission in central China.</p><p><b>METHODS</b>(1) Cross-sectional study: couples that one was HIV positive were selected in Henan and Hebei province of China. The couples must be 20 - 50 years old with normal function on sexual intercourse. Cordant couples that subsequently infected partners were at risk of infection solely through sexual contact with the HIV-seropositive partner and the discordant couples that the seronegative partners were at risk of infection solely through sexual contact with the HIV-seropositive partner, were selected. Plasma viral load, CD4 cell count were tested. (2) Case-control study was used to compare 7 sexual transmitted cases and 56 nontransmitted controls with respect to the frequency of sexual intercourse, plasma viral load and CD4 cell count.</p><p><b>RESULTS</b>(1) A total of 87 couples that at least one partner was HIV positive were recruited include 56 discordant couples and 7 cordant couples with whom sexual transmission had happened. The rate of sexual transmission was 11.1% among those at the risk of sexual transmission. (2) Of the discordant couples, male positive rate 25%, female positive was 75%. (3) The risk for transmission was higher in those couples with the frequency of unprotected vaginal sexual intercourse (> or = 4 times per month) than the reference group (< 4 times per month) (Fisher's exact test, P = 0.047, OR = 8.0). Median plasma viral load was significantly higher in the antecedent infected partners of cordant couples than the positive partner of discordant couples (378,285.71 vs 136,578.57 copies/ml, t = 3.591, P < 0.01). The odds ratio was 22.0 for plasma viral load > or = 100,000 copies/ml compared with the reference group of < 100 000 copies/ml (Fisher's exact test, P = 0.016). The CD4 cell count and CD4/CD8 of the transmitted group were significantly lower than that of the nontransmitted (t = 2.767, P < 0.05; t = 6.06, P < 0.05).</p><p><b>CONCLUSIONS</b>The frequencies of heterosexual-transmission in central China were relatively low. The risk of heterosexual transmission was related to the frequency of sexual intercourse. Higher plasma viral load and lower CD4 count was strongly correlated with high risk of heterosexual transmission.</p>