Analyses Of HIV-1 Subtypes And Primary Drug Resistance Mutations In Yunnan Border Areas

ObjectiveDetection of subtypes and primary drug resistance（PDR）of human immunodeficiency virus type 1（HIV-1）helps to understand HIV epidemic and its dynamic changes,treat HIV/AIDS patients,and control HIV epidemic,which is of important public health and clinical significance.There have been some research reports about HIV-1 subtypes and PDR in some of Yunnan border areas,which still lack a holistic picture of the issue.It is of great significance to comprehensively investigate HIV-1 subtypes and PDR in Yunnan border areas.The aim of this study was to investigate subtypes and distributions of HIV-1 and primary drug resistance mutations（PDRMs）among newly diagnosed HIV-infected persons in Dehong Dai and Jingpo Autonomous Prefecture（Dehong Prefecture）,Xishuangbanna Dai Autonomous Prefecture（Banna Prefecture）,and Honghe Hani and Yi Autonomous Prefecture（Honghe Prefecture）in Yunnan Province,which shares its border with Myanmar,Laos,and Vietnam;and to compare the data between Chinese and foreign HIV-infected persons,so as to provide evidence for HIV/AIDS prevention,control and treatment in border areas of Yunnan Province.MethodsFrom November 1,2015 to October 31,2016,150 newly diagnosed HIV-infected individuals from Myanmar,Laos or Vietnam were continuously enrolled in Dehong,Banna and Honghe Prefectures in the border areas of Yunnan Province by means of screening,cross-border testing and hospital laboratory testing,while another 150 Chinese adult citizens were involved in the study who were diagnosed with HIV infection in the same time period and the same county/city in Dehong,Banna,and Honghe Prefectures,Yunnan Province of China.All of the participants in the study were investigated under the informed consent.Their blood samples were collected by local trained professionals from centers for disease control and prevention（CDC）,and the subjects were interviewed in a face-to-face manner,in which data were collected on demographic information and HIV infection related information.HIV-1 RNA was extracted from the blood sample,and then reverse transcription polymerase chain reaction（RT-PCR）was carried out for complementary DNA（cDNA）before partial env and pol fragments were amplified with nested PCR.HIV-1 subtypes and primary drug resistance mutations were detected throught electrophoresis identification,sequencing and sequence analysis of PCR amplification products.Primary drug resistance mutations were identified by using the Surveillance Drug Resistance Mutation（SDRM）Worksheet（2014）in HIV Resistance Database of Stanford University,and an application（APP）based on the HIV Resistance Database of Stanford University was used to detect types of resistant drugs and resistance level.Chi-squared tests were used to study the relationship between primary drug resistance and HIV-1 genotypes;to investigate the relationship between HIV-1 genotypes,primary drug resistance and demographic characteristics,ways of finding HIV infection,transmission routes,multiple sexual partners,and condom use during the whole sexual activity;and to explore the relationship between multiple sexual partners,condom use during the whole sexual activity and nationality,and transmission routes.Logistic regression models were employed to analyze whether demographic characteristics,ways of finding HIV infection,transmission routes,multiple sexual partners,and condom use during the whole sexual activity were associated with HIV-1 genotypes and primary drug resistance.A value of P<0.05 was set as statistically significant.Results1.Multiple sexual partners and condom use among newly reported HIV-infected persons in Yunnan border areas1.1 Of the 300 newly reported HIV-infected people,256 responded to a question about the number of their sexual partners six months before they were diagnosed with HIV infection.As a result,23.8%（61/256）had multiple sexual partners.The proportions of Chinese and foreign infected people with multiple sexual partners were 18.9%（25/132）and 29.0%（36/124）,respectively,with no statistical difference between them（χ2=3.588,P=0.058）.1.2 Of the 300 newly reported HIV-infected people,208 responded to a question on condom use six months before they were diagnosed with HIV infection.As a result,25.5%（53/208）of them used condoms during the whole sexual activity.The percentages of Chinese and foreign infected people using condoms during the whole sexual activity were 36.2%（38/105）and 14.6%（15/103）,respectively,with a statistical difference between the two groups（χ2=12.808,P=0.000）.2.Genotypes of HIV-1 in Yunnan border areas2.1 The majority of subtypes of HIV-1 in Yunnan border areas included CRF01_AE（30.0%,68/227）,Unique Recombinant Forms（URFs）（26.9%,61/227）and CRF08_BC（21.6%,49/227）,while other genotypes included subtype C（10.1%,23/227）,CRF07_BC（6.2%,14/227）,subtype B（3.1%,7/227）,CRF55₀1B（1.3%,3/227）,subtype A（0.4%,7/227）and CRF64_BC（0.4%,7/227）.URFs mainly included B/C（15.0%,34/227）,CRF01_AE/C（4.8%,11/227）,CRF01_AE/B/C（4.4%,10/227）,and CRF01_AE/B（2.6%,6/227）.2.2 The HIV-1 genotypes and their distributions in Chinese and foreign nationals were different.Nine genotypes of HIV-1 were detected in Chinese infected persons,including CRF08_BC（32.1%,34/106）,CRF01_AE（22.6%,24/106）and URFs（20.8%,22/106）.Six genotypes of HIV-1 were determined in foreign infected persons,including CRF01_AE（36.4%,44/121）,URFs（32.2%,39/121）and subtype C（14.0%,17/121）.The distributions of HIV-1 genotypes among Chinese and foreign nationals showed a significant difference（X2=31.526,P=0.000）.2.3 Genetic diversity of HIV-1 strains was observed in Dehong border area with URFs（39.5%,58/147）being the most prevalent subtype.The most prevalent strain was CRF08_BC（55.2%,16/29）in Banna Prefecture and CRF01_AE（60.8%,31/51）in Honghe Prefecture.Statistical differences were observed in the distributions of HIV-1 subtypes between Dehong and Banna Prefectures（χ2=45.396,P=0.000）,and between Dehong and Honghe Prefectures（χ2=57.505,P=0.000）,but no statistical difference between Banna and Honghe Prefectures（χ2=8.481,P=0.050）was observed.2.4 In Dehong Prefecture,URFs（28.3%,13/46）and CRF07_BC（19.6%,9/46）were the main genotypes among the Chinese people infected via heterosexual transmission.URFs（31.4%,16/51）and CRF01_AE（31.4%,16/51）were the major genotypes in people from Myanmar who were infected through heterosexual transmission.There was a statistically significant difference in the genotype distributions between Chinese and Burmese heterosexually transmitted individuals（χ2=18.490,P=0.005）.2.5 Compared with other genotypes（including subtype B,subtype C,CRF07_BC,CRF55₀1B,CRF64 BC,and subtype A）,Chinese infected persons were less likely to carry a CRFO1_AE virus than foreign infected persons（Chinese nationality:OR=0.392,95%CI:0.167-0.920）,workers infected with HIV were less likely to carry a CRF08_BC virus than infected people with other occupation（workers:OR=0.073,95%CI:0.006-0.831）,and people infected via heterosexual transmission were less likely to carry a URFs virus than those infected via intravenous drug use（heterosexual transmission:OR=2.653,95%CI:1.016-6.927）.3.HIV primary drug resistance in Yunnan border areas3.1 Primary HIV drug resistance rate in Yunnan border areas was 9.5%（17/179）,among which the primary drug resistance rate in Chinese infected persons was 13.6%（11/81）,while that of foreign infected persons was 6.1%（6/98）.The primary drug resistance rates in both groups were at a moderate level（5.0%-15.0%）,with no significant difference between them（χ2=2.870,P=0.090）.3.2 The prevalence of surveillance drug resistance mutations（SDRMs）was 8.8%（10/114）,7.4%（2/27）,and 13.2%（5/38）in Dehong,Banna,and Honghe Prectures,respectively,which was all at a moderate level（5.0%-15.0%）with no significant difference observed（χ2=0.866,P=0.751）.3.3 Proportion of drug-resistant strains in Chinese HIV-infected persons（15.9%,7/44）was bigger than that（4.3%,3/70）in Burmese in Dehong Prefecture with a statistically significant difference observed（χ2=4.561,P=0.044）.3.4 Primary drug resistance mutations included mutations to three kinds of antiviral medications.The resistance mutations to NNRTIs included K103N,Y181C,K101E,G190A,V106M and P225H.K103N and V106M cause high-level resistance to EFV and NVP.Y181C and G190A confer high-level resistance to NVP.P225H causes intermediate-level resistance to EFV and NVP.G190A causes intermediate-level resistance to EFV.The resistance mutations to NRTIs were M184V,K65R,T215S,V75M,L74I,and K219Q.M184V confers high-level resistance to FTC and 3TC.K65R causes high-level resistance to TDF,but intermediate-level resistance to ABC,FTC and 3TC.The resistance mutations to PIs were L24I,L76V,L90M and M46I.Mutations with high-level resistance to ATV/r,DRV/r or LPV/r were not observed.3.5 Eleven categories of drug resistance mutations were found in Chinese infected persons,which were more than those detected in foreign infected persons.Among the mutations,K103N,P225H,V106M,and K101E were resistant to NNRTIs;M184V,L74I,K65R,and K219Q were resistant to NRTIs;L24I,L76V,and L90M were resistant to PIs.There were 6 drug resistance mutations in foreign infected persons,among which K103N,Y181C,and G190A were resistant to NNRTIs;T215S and V75M were resistant to NRTIs;M46I was resistant to PIs.K103N（NNRTIs-resistant）was the drug-resistant mutation found in both Chiese and foreign groups,which was highly resistant to EFV and NVP,and the rest mutations were only found in either group.3.6 Subtypes and their proportions of those with drug resistance were CRF55₀1B 33.3%（1/3）,CRF07_BC 20.0%（1/5）,CRF01_AE/B 20.0%（1/5）,CRFO1_AE 16.7%（9/54）,B/C 8.8%（3/34）,and CRF08_BC 7.7%（2/26）,respectively.For the Chinese infections,the subtypes and their drug resistance proportions were CRF01_AE/B 50.0%（1/2）,CRF55₀1B 33.3%（1/3）,CRF07_BC 25.0%（1/4）,CRF01_AE 21.1%（4/19）,B/C 12.5%（2/16）,and C RF08_BC 9.1%（2/22）,respectively.For the foreign infections,the subtypes and their drug resistance proportions from large to small were CRF01_AE 14.3%（5/35）and B/C 5.6%（1/18）,respectively.3.7 During six months prior to learning of HIV infection,three（17.6%）of the 17 primary drug-resistant individuals had multiple sexual partners,among whom two were Chinese and one was foreigner;eight（47.1%）of them experienced sexual activities without condoms,among whom half were Chinese and half were foreigners.Five（29.4%）of those with high-level drug resistance mutations had sexual activities without condoms,among whom two were Chinese and three were foreigners.3.8 Primary multidrug resistance was detected.Mutations resistant to NRTIs（K65R and M184V）and NNRTIs（K103N and P225H）were detected in a Chinese HIV-infected individual.A Vietnamese HIV-infected individual was found to carry mutations resistant to NRTIs（V75M）and PIs（M46I）.Conclusion1.High risk of spreading HIV from HIV-infected people before HIV infection is diagnosed in Yunnan border areasBefore people infected with HIV in Yunnan border areas know their infection status,the proportion of multiple partners is relatively high,and proportion of condom use in the whole sexual activity is low,which implies a high risk of HIV spread.In Yunnan border areas,infected foreigners have a lower proportion of condom use in the whole sexual activity than the Chinese infected people although both groups share the low condom use proportion.2.Diversity of HIV-1 genotypes was found in Yunnan border areas with complex characteristics in their distributions2.1 There are more HIV-1 genotypes among Chinese infected persons than foreign infected persons in Yunnan border areas,including CRF08_BC and CRF07_BC,which are prevalent in the eastern and central parts of Yunnan Province,and CRF55₀1B,which is prevalent in men who have sex with men（MSM）in eastern China.2.2 The proportion of URFs in Dehong border area is high,in both Chinese and foreign HIV-infected people regardless of infection via heterosexual transmission or intravenous drug use,similar to the high proportion of URFs in northern Myanmar.2.3 CRFO1_AE is most prevalent in the border area of Honghe Prefecture.It is the most prevalent among Vietnamese infections,and the second prevalent among Chinese infections.CRP01_AE is almost as prevalent as CRF08_BC in Chinese infections.It reflects that the border area of Honghe Prefecture and neighboring Vietnam have a trend of convergence in the spread of HIV.2.4 The proportion of MSM in Banna Prefecture is relatively high,and the proportion of CRF08_BC in the group is higher than that in MSM in other areas,suggesting that MSM in Banna may be relatively isolated.3.The prevalence of SDRMs is at a moderate level in Yunnan border areas3.1 The primary drug resistance rates are at a moderate level（5%-15%）in Dehong,Banna,and Honghe Prefectures.3.2 More than half of those with HIV primary resistance mutations are highly resistant to EFV and/or NVP in NNRTIs.4.It is necessary to further strengthen HIV prevention and control in Yunnan border areas4.1 Only 25.5%of HIV-infected people used condoms in their sexual life six months before they knew they were infected with HIV,which is a high risk of transmission of HIV with drug resistance mutations,suggesting the need to further strengthen HIV prevention and control education and promote condom use in the general population.4.2 Some foreign infections in Yunnan border areas also have the risk of transmission of primary drug resistance mutations,and it is necessary to strengthen cooperation with relevant departments of neighboring countries for joint management and control of HIV infection.4.3 Seventeen point six percent of HIV-infected people with primary drug resistance mutations had multiple sexual partners and 47.1%did not use condoms in their sexual activities six months before they knew they were infected with HIV.Therefore,it is imperative to explore effective approaches to screening primary drug resistance,to manage HIV/AIDS patients with drug resistant mutations in an individualized manner,and to provide antiviral treatment in the early stage in order to further strengthen HIV prevention.4.4 It is necessary to be alert to the risk of high-level primary resistance to EFV and NVP in patients for their first antiretroviral therapy.Drug resistance testing can be carried out wherever it is feasible before antiviral treatment to guide clinical use of drugs.