Research On The Epidemiological Characteristics And Molecular Evolution Of HIV/AIDS Cases Among Young People Newly Diagnosed In Nanjing

ObjectivesHIV/AIDS is still a major global public health problem.Although AIDS epidemic is generally in a low-prevalence situation in China,the number of young people infected with HIV has increased rapidly in recent years,and has gradually become a key population for the prevention and control of the AIDS epidemic.This study analyzed the epidemic status and molecular evolution characteristics of newly diagnosed HIV/AIDS cases among young people in Nanjing by epidemiological investigation and molecular phylogeny,and further understood the epidemic dynamics and evolutionary laws of HIV-1 strains,to provide a scientific basis for the prevention and control of AIDS for young people in Nanjing.Methods1.Cross sectional study:one-to-one survey conducted among 744 young HIV/AIDS cases newly diagnosed in the five district-level Centers for Disease Control and Prevention in Nanjing,including Xuanwu,Qinhuai,Gulou,Qixia,and Jiangning from September 2015 to June 2019,to collect basic information about cases,AIDS-related knowledge and sexual behavior information.To undstand the basic demographic characteristics of the population and AIDS-related knowledge and behaviors.2.Research on HIV molecular epidemiological characteristics:The whole blood samples of the research subjects were collected simultaneously during the questionnaire survey.Through PNA extraction,RT-PCR,nested PCR,sequencing,and splicing to obtain HIV-1 pol region gene fragments（1060 bp pol region gene（covering the protease region 1～99 amino acids and part of the reverse transcriptase region 1～254 amino acids）,HXB2:2253～3312）.An approximate maximum likelihood tree was constructed to determine the HIV-1 genetypes;the Stanford University HIV drug resistance database was used to analyze drug resistance mutation sites;ClusterPicker was used to screen drug resistance transmission clusters,there were inclusion criterias:cluster node value（bootstrap）≥ 0.9 and maximum gene distance within cluster（GD）<0.03 nt substitution per site.3.HIV phylogenetic analysis:Downloaded the reference sequence from the Los Alamos HIV Database（LANL,www.hiv.lanl.gov）database,merging it with the Nanjing sequences successfully amplified by our laboratory into a new database,constructed a phylogenetic tree to describe different subtypes evolutionary characteristics of strains.Infer the evolution rate and time origin of the virus strain based on the software package of BEAST;infer the epidemic trend by reconstructing the effective population size of different subtypes by using the Bayesian skyline diagram.4.Statistical analysis:SPSS24.0（SPSS Inc.Chicago,IL,USA,2015）software was used for statistical analysis.The quantitative variable conformed to the normal distribution are described by the mean±standard deviation（x±s）,and the t-test is used for comparison between groups;the count data is described by the constituent ratio or rate（%）,and the χ2 test is used for comparison between groups.P<0.05 indicated that the difference is statistically significant.Results:1.Basic information:A total of 744 young HIV/AIDS cases were investigated,including 240 young students and 504 non-young students.Mainly male,accounting for 96.1%（715/744）,and the main route of infection was homosexual sex;compared with non-students,young students had a higher proportion of homosexual infections and a higher education level（P<0.001）,A smaller age at diagnosis and a lower proportion of foreign household registration（P<0.001）.2.AIDS-related knowledge.The average score of AIDS-related knowledge among students in this study was（6.8±1.2）,and the overall awareness rate was 84.6%;the average score of non-students’AIDS-related knowledge was（7.6±1.3）,and the overall awareness rate was 94.8%;The awareness rate of all the items were higher than 70%among no-students,while the awareness rate of some items of young students was lower than 70%.3.AIDS-related sexual behavior.The age of first sex was（18.9±2.0）years old among students was lower than the age of first sex of non-students（20.8±2.3）years old.Compared with non-students,the students had a lower age of first sex（P<0.001）;Unprotected anal sex and risky sexual behaviors with multiple partners were common in young population,and the types of sexual partners were diversified.There were differences in the distribution of types of sexual partners between students and non-students（P<0.001）.4.HIV-1 genotypes distribution.704 pol gene fragment sequences were successfully amplified from 744 blood samples（94.6%,704/744）.The HIV-1 subtypes analysis revealed 10 different HIV-1 genotypes,of which the main genotype was CRF01_AE（42.6%,300/704）and CRF07_BC（31.3%,220/704）,followed by URFs,CRF67₀1B,B subtype,CRF68₀1B,CRF55₀1B,CRF08_BC and CRF59₀1B,accounted for 11.1%（78/704）,4.1%（29/704）,3.7%（26/704）,3.4%（24/704）,2.7%（19/704）,0.7%（5/704）and 0.4%（3/704）,respectively.5.HIV-1 drug resistance.In this study,22 kinds of drug resistance mutations were found,including 4 kinds of protease（Protease,PR）gene mutations and 18 kinds of reverse transcriptase（Reverse Transcriptase,RT）gene mutations.The total drug resistance rate was 14.5%（102/704）,and the rate of transmitted drug resistance mutations was 4.5%（32/704）.There was no statistical difference between the total drug resistance rate and the transmitted drug resistance rate between students and non-students（P>0.05）.There were 10 drug-resistant transmission clusters formed in the drug-resistant transmission network,which mainly include V179/E/T,M46I and E138G mutation sites.6.HIV-1 epidemic characteristics.CRF01_AE,CRF07_BC and CRF67₀1B were the three most prevalent HIV-1 strains among young people in Nanjing.CRF₀1AE has formed 5 evolutionary clusters in this population,among which Cluster2,Cluster4 and Cluster5 corresponded to the main popular Chinese CRF01_AE ClusterⅣ-4a,ClusterⅣ-4b and Cluster Ⅴ evolutionary clusters respectively;CRF₀7BC forms a larger pedigree and a smaller evolutionary cluster Cluster;CRF67₀1B formed two evolutionary clusters.HIV/AIDS cases in the evolutionary cluster were mainly infected by men who hadsex with men,but also included a certain proportion of people who have been infected by heterosexual sex.7.HIV-1 evolution analysis.The most common ancestor times of CRF01_AE,CRF07_BC and CRF67₀1B were 1984.07（1979.25,1988.09）,1993.16（1989.90,1996.88）and 2006.03（2003.25,2007.70）,and the evolution rates were 2.06×10-3（1.78×10-3,2.31×10-3）、1.80×10-3（1.56×10-3,2.05×10-3）and 1.4×10-3（1.1×10-3,1.8×10-3）nt substitutions/site/year.The BSP chart showed that the effective population size of these three subtypes had finally stabilized after a certain increase and change in the early stage.Conclusions1.MSM was a key population for the prevention and control of the AIDS epidemic among young people.This population was relatively open-minded and active in sexual behaviors.Unprotected anal sex,multiple sexual partners,and multiple types of sexual partners were common.Although young students were highly educated,their awareness of AIDS-related knowledge is low.Therefore,schools should systematically carry out sex education to improve the level of AIDS-related knowledge of young people.And explore new educational model to promote the transformation of health knowledge into healthy behaviors and reduce the spread of HIV.2.The HIV-1 epidemic strains in the young population in Nanjing were diverse and complicated,especially new strains and unique recombinant strains account for a relatively high proportion;the total drug resistance mutation rate was 14.5%,and the transmission resistance rate was 4.5%,which was close to the warning level of 5%.In addition to traditional epidemiological investigations,future HIV-1 prevention and control efforts should also focused on molecular epidemiological research,strengthened the surveillanceof subtypes and drug-resistant mutations in the young population,and fully understood the characteristics and epidemic dynamics of HIV-1.3.CRF01_AE,CRF07_BC and CRF67₀1B were the main epidemic strains among young people in Nanjing.These strains gradually evolved to form two or more evolutionary clusters,with new epidemic characteristics.The evolutionary tree showed that men who have sex with men were closely related to people of heterosexual crowds,and there was a risk of cross-infection.The BSP chart showed that these three subtypes had formed a stable effective population size,it may cause a wider epidemic.