HIV-1 Molecular Epidemiology And Drug Resistance In Guangxi And Northern Vietnam

Part 1 HIV-1 Molecular Epidemiologyin Guangxi and Northern VietnamBackground and ObjectiveGuangxi is one of HIV-hit areasin China,with a relatively high HIV/AIDS epidemic.The northern Vietnam region bordering Guangxi is also a relatively serious HIV/AIDS epidemic in Vietnam.Previous studies have shown that HIV-1 strains may be transmitted to each other between Guangxi and northern Vietnam.However,most of these molecular epidemiology studies have only used the HIV epidemic strains collected locally and compared with published HIV-1 strains from China or Vietnam,respectively.In recent years,the transmission routes and major subtypes of HIV-1 in Guangxi and northern Vietnam have changed.So far,there has not been a large-scale epidemiological study conducted at the same time in both regions to clarify whether there is a transmission relationship between HIV-1 strains in the two places.At present,the political,economic,and cultural exchanges between Guangxi and Vietnam have become increasingly active along with the implementation of the―Belt and Road Initiative‖and the increasingly close China-ASEAN cooperation,and the risk of HIV-1 spreading to each other has also increased.In this context,the main purpose of this study was to analyze HIV-1 subtype distribution,genetic variation,and phylogenetic evolution in both regions,in order to understand HIV-1 prevalence characteristics,genetic variation and mutual transmission.MethodsIn 2006,through convenient sampling,HIV-infected people being in antiviral therapy from 6 cities from Guangxi（including Chongzuo City,Fangchenggang City,Nanning City,Liuzhou City and Baise City）and 4 border provinces from northern Vietnam（Quang Ninh Province,Liangshan Province,Gaping Province,and Hejiang Province）as research subjects.Questionnairewas used to conduct epidemiological investigation on the subjects to obtaine basic demographic data and HIV-related behaviors,infection routes,infection time,treatment status,and cross-border behaviors.Ten ml of venous blood was collected and peripheral blood mononuclear cells（PBMCs）were isolated.Previral DNA was extracted from cells,and HIV-1 gag,pol,env C2V3 gene fragments were amplified by Nest-PCR.The positive amplified products were then sequenced and edited using Bioedit and MEGA software The edited gene sequences were submitted to the HIV database for subtype analysis and recombination analysis.At the same time,MEGA,Simplot and other bioinformatics softwares were used to improve and confirm the subtypes,recombinationbreakpoint,geneticvariation,andphylogenetic evolution.Furthermore,the sequences were submitted to the website（http://www.atgc-montpellier.fr/）to build the ML tree.Finally,data were analyed by using Spss 23.0 software.Results1.Sample collection and HIV-1 gene amplificationA total of 1000 HIV-1-infected patients were enrolled in the study.Among them,567 patients were from Guangxi,and 433 patients were from Vietnam.Totally,we obtained 613gene sequences from 1000 subjects,including 399 in Guangxi and 214 in northern Vietnam.As for different genes,200 gag sequences,399pol sequences,and 178 env sequences were successfully amplified fromGuangxi;112 gagsequences,214 polsequences and 140 env sequences were obtained from Northern Vietnam.2.HIV-1subtype determination and distributionThe subtypes of 613sequences were identified,including 234CRF01_AE,106 CRF08_BC,54 CRF07_BC,1 B,1 C and 3 URFs in Guangxi,as well as 214 CRF01_AE were identified in Northern Vietnam.3.The distribution of HIV-1subtypes in transmission routeThe distribution of HIV-1subtypes in the heterosexual transmission（HST）population in Guangxi was as follow:CRF01_AE accounting for 34.09%（136/399）,CRF08_BC accounting for 4.51%（18/399）,and CRF07_BC accounting for 1%（4/399）.The distribution of HIV-1 subtypes in injection drug users（IDUs）in Guangxi was 21.8%（87/399）CRF01_AE,19.8%（79/399）CRF08_BC,and 11.3%（45/399）CRF07_BC.The CRF01_AE subtype accounted for 39.9%and 47.7%in heterosexual transmission route and injection drug use route,respectively,in Northern Vietnam.4.ML phylogenetic tree analysis of CRF01_AE strainsThe CRF01_AE strains in Guangxi and northern Vietnam can be divided into five evolution clusters.Among them,IDUs and few HETs in Liuzhou City of Guangxi were clustered into clusters 1;HETs and few IDUs in Guangxi border cities as well as IDUs and HETs in northern Vietnam were clustered into2;Nanning’s four IDUs and Fangchenggang’s one HET clustered into cluster3;A small part of IDUs and HETs in Guangxi and a small part of IDUs and HETs in northern Vietnam were independently clustered into two independent clusters（VC1 and VC2）.HIV-1 strains among IDUs in northern Vietnam may spread to some HETs in Guangxi border cities.5.Analysis of unknown recombinant subtypesThree HIV-1 strains were identified as unknown recombinant subtypes.The recombinant patterns were CRF01_AE/B（B’）and CRF01_AE/B（B’）/C.6.The genetic distance analysisThe genetic distances of gag,poland envgenes and their coding regions P24,P17,P51,P15 and P31 in CRF01_AE subtype from Guangxi were significantly higher than those from northern Vietnam（p<0.01）.The genetic distance in env region of CRF01_AE subtype in Guangxi and northern Vietnam were greater than that in pol region,and the gene distance in pol region was greater than that in gag region,with a statistically significant difference（p<0.05）.In gag coding region,the genetic distance of P17 was higher than that of P24 among all CRF01_AE subtypes,whereas in pol coding region,from high genetic distance to low one,the order is P51,P15 and P31.According to the genetic distance of env,the epidemic timeof CRF01_AE subtype in Guangxi is estimated to be about 15¹7 years,and the epidemic time in northern Vietnam is about 9¹1 years.7.The p6△7 deletion in three recombinant CRF07_BC strains.A unique deletion of seven amino acid（aa）（p6△7）（aa 30–36,PIDKELY,in HXB2 region）was observed in three recombinant CRF07_BC strains in Guangxi.Conclusions1.In Guangxi,HIV-1-infected patients were mainly transmitted through heterosexual contact and injecting drug use.SubtypesCRF01_AE,CRF08_BC,and CRF07_BC co-existed in Guangxi.In northern Vietnam,HIV-1-infected patients were mainly transmitted through heterosexual contact and injecting drug use,and the main subtype was CRF01_AE.2.HIV-1 strain among IDUs in northern Vietnam may be transmitted to the HETs in Guangxi border City.HETs of Guangxi and IDUs and HETs of Vietnam have a spreading relationship.IDUs in northern Guangxi had no relationship with IDUs in northern Vietnam.3.Unknown recombinant subtypes CRF01_AE/B（B’）and CRF01_AE/B（B’）/C found in Guangxi might be the recombination of CRF01_AE and CRF07/08_BC.Part 2 HIV-1 drug resistance and the influencing factors among HIV-1/AIDS patients in Guangxi and Northern VietnamBackground and ObjectiveWith the global antiviral treatment,HIV-1 resistance has become an serious issue.The HIV-1 CRF₀1AE and CRF₀7/08BC subtypes epidemic in Guangxi,while CRF₀1AE is the main subtype in Vietnam.Due to the different characteristics of drug-resistant strains of different subtypes,and different ART programs in different countries.The occurrence and development of HIV-resistant strains in Guangxi and northern Vietnam should have their own characteristics.Although there have been many studies reported HIV-1resistance and influencing factors in China and Vietnam.However,no studies have yet revealed the differences of HIV resistance between Vietnam and China,as well as the factors affecting drug resistance at the China-Vietnamborder.Thus,the main purpose of this part of study was to study the incidence of drug resistance,drug resistance mutations,and drug-resistant populations,as well asfactors affecting the HIV-1 drug resistance in treatment HIV/AIDS patients in Guangxi and northern Vietnam by collecting samples from both regions.The study will greatly help to monitor the prevalence of drug resistant HIV-1 strains in Guangxi and Northern Vietnam as well as to establish rational antiviral therapy program for HIV/AIDS patients.MethodsThe 613 pol gene sequences obtained from the first part of this study were submitted to HIV Drug Resistance Database at Stanford University（http://hivdb.stanford.edu）,to get the resistance mutation and score results.The database about the demographic information of all subjects was established by EpiData software.Spss23.0 statistical software was used for descriptive statistical analysis.Comparison Mean was performed using t-test or analysis of variance.Drug resistance rates between different groups were compared using Chi-square test or Fisher’s exact probability method.Influencing factor analysis was performed by chi-square test and logistic regression.Results1.A total of 613 pol gene sequences were effectively amplified and successfully sequenced,including 399 sequencesfrom Guangxi,of which 46were resistant to drugs,with a drug-resistance rate of 11.53%（46/399）,as well as214pol sequences from Vietnam,of which 21 were resistant todrugs,with a drug-resistance rate of 9.81%（21/214）.There was no significant difference between Guangxi and northern Vietnam in drug resistance rate（χ²=0.421,p=0.516）.2.There is no significant difference in the drug resistance rate among different cities in Guangxi（χ²=1.48,p=0.0.915）,as well as different provincesin northern Vietnam（χ²=6.4,p=0.94）.3.The differences between Guangxi and northern Vietnam in low,medium and high resistances were not statistically significant,respectively.In Guangxi and northern Vietnam,the high resistanceaccounted for 7.5%（30/399）and 7.9%（21/214）,respectively.4.The drug resistance rate of subtype CRF01_AE,CRF07_BC,CRF08_BC in Guangxi was 11.4%,12.96%,11.32%,respectively.All the drug resistances in northern Vietnam were found in CRF01_AE subtype,and the drug resistance rate was 9.81%.The drug resistance rate of CRF01_AE between Guangxi and northern Vietnam was not statistically significant.5.The mutation rate of NNRTIs of HIV-1 strains in Guangxi was 5.76%,which was higher than that in Northern Vietnam（1.87%）,and the difference was statistically significant（χ²=5.019,p=0.025）.Among all the drug-resistant cases,in Guangxi,the composition ratio of NNRTIsis 50%,which is higher than that in northern Vietnam（19.05%）,and the difference was statistically significant（χ²=6.09,p=0.014）.The composition ratio of dual drug resistance（NRTIs+NNRTIs）（32.61%）in Guangxi was lower than that in northern Vietnam（66.6%）,and the difference was statistically significant（χ²=6.459,p=0.011）.6.In Guangxi,the prevalence of DRMs against NRTIs,NNRTIs,PIs,was1.00%（4/399）,5.76%（23/399）,1.00%（4/399）,respectively.And 15 patients（3.76%,15/399）were resistant to both NRTIs and NNRTIs.7.In northern Vietnam,the prevalence of DRMs against NRTIs,NNRTIs,PIswas 0.93%（2/214）,1.87%（4/214）,0.47%（1/214）,respectively.And 14patients（6.54%）were resistant to both NRTIs and NNRTIs.8.In Guangxi,M184V was the most frequently found resistant mutation against NTIRs,while K103N,M230I/L,G190A/E,and E138A/K were the frequent mutations against NNTIRs.I54L and V82V/F were the main mutations against PIs in Guangxi.9.In northern Vietnam,M184V/I,V75M,K65R were the frequent mutations against NTIRs,while Y181C,G190A/R/S,K103N were the main mutations against NNTIRs.N88S,L89V,L90M,L10F/I were the main mutations against PIs in northern Vietnam.10.There were significant differences in mutations at positions 181 and65 in Guangxi and northern Vietnam（χ²=8.514,p=0.004 andχ²=5.940,p=0.015）.11.In Guangxi,the rate of moderate resistance to ETR（NNRTIs）was1.75%,which was lower than that in northern Vietnam（4.467%）,and the difference was statistically significant（χ²=4.4,p=0.036）.In Guangxi the rates of resistance to ABC,D4T and DDI（NRTIs）were 0.5%,0.25%and 0.25%,respectively,which are lower than those in northern Vietnam（3.27%,3.74%,4.67%,respectively）,and the difference was statistically significant（p<0.05）.12.Multivariable logistic regression revealed that the rates of DRMs were significantly higher among patients aged 15-24（OR=19.87;95%CI:1.80-220.20）and those receivedthe treatment regimen containing EFV（OR=4.43;95%CI:1.22-16.07）or LPV/r（OR=8.08;95%CI:1.73-37.84）.Conclusions1.The overall drug resistant rate is low in both Guangxi and northern Vietnam.In Guangxi,drug resistant mutation was mainly the mutations against NRTIs;Innorthern Vietnam,dual resistant mutations（against NRTIs and NNRTIs）were more popular.In both areas,the occurrence of mutations against PIs was seldom.2.The M184V/I mutation was a hot spot generated in NRTIs in northern Guangxi and northern Vietnam,and the K103N and G190A/E mutations occurred in the NNRTIs.The resistance mutations generated in PIs were different between these two areas.3.Drug-resistant patients in Guangxi mainly developed resistance to 3TC,ABC,FTC,EFV,and NVP,while drug-resistant patients in Vietnam mainly developed resistance to 3TC,ABC,D4T,FTC,TDF,EFV,and NVP.4.Age and medication regimens containing EFV or LPVr are risk factors for the occurrence of drug resistance.