Surveillance of Transmitted Drug Resistance in HIV-1-Infected Youths Aged 16 to 25 Years,a Decade After Scale-up of Antiretroviral Therapy in Hebei,China

Abstract

Sexual exposure has been the predominant route of HIV-1 spread in Hebei Province, China. However, little information is available on HIV-1-transmitted drug resistance (TDR) among HIV-1-infected youths aged 16 to 25 years who are infected with HIV-1 and sexually active. In this study, the overall prevalence of TDR was 6.6% (10/152), a moderate level (5.0%–15.0%) according to World Health Organization Threshold Survey guidelines. However, the prevalence of TDR to protease inhibitors and nonnucleoside reverse transcriptase inhibitors was 4.6% (7/152) and 2.0% (3/152), respectively, which correspond to a low level (<5.0%). All TDR mutations (M46L/I, Y181C, K101E, and G190E) were found only in youths infected with HIV-1 through sexual activity. The prevalence of TDR among heterosexuals (10.0%, 3/30) was higher than that among men who have sex with men (5.8%, 7/120). TDR mutations resided in CRF01_AE (M46I/L and G190E) and subtype B (Y181C and K101E). Our findings will provide useful information on which drug regimens to use in treating the newly infected people with HIV-1.

The human immunodeficiency virus type 1 (HIV-1) epidemic has been a major global public health problem for over 30 years. Countries worldwide that have been affected by HIV-1 have made great efforts to prevent and control the spread of the virus. Antiretroviral therapy (ART) has dramatically extended the lifespan of patients infected with HIV.¹ However, HIV-1 has high genetic variability and rapid viral replication,² which cause transmitted drug resistance (TDR) to the antiretroviral drugs used in ART.^1,3 TDR can exhaust the choices of antiretroviral drugs and therapeutic regimens, accelerating the development of AIDS and increasing mortality in patients with HIV/AIDS.⁴

Hebei Province neighbors Henan Province at its southern border and encompasses Tianjin and Beijing. The first case of HIV-1 infection in Hebei was identified in 1989 among workers returning from Africa.⁵ In the 1990s, Hebei was defined as an area where most HIV-1 infections were caused by commercial blood products contaminated by HIV.⁶ However, the exposure owing to high-risk sexual behavior has become the predominant driver of a trend that has been increasing since 2005 of newly diagnosed HIV-1 infections in the province.⁷

Free ART was launched in 2003 for Hebei Province. The national guidelines for ART have changed over the years from treating patients with CD4 < 200, to CD4 < 350 in 2011, to now treating all HIV-diagnosed individuals in China with ART.

To date, ART has been implemented for a total of 10 years and has improved patient quality of life.⁸ However, a high prevalence of HIV-1 drug resistance (DR) has been observed among patients receiving ART in Hebei.⁹ Low prevalence of TDR has also been identified among recently HIV-infected men who have sex with men (MSM) at MSM sentinel surveillance points.⁷ With the scale-up of ART, TDR surveillance will provide useful information on which drug regimens to use in treating people newly infected with the virus. Moreover, sequences and phylogenetic analyses can provide much evidence about how HIV is spreading in the population. The objective of our study was to investigate the prevalence of TDR among newly diagnosed, treatment-naive young people, aged 16 to 25 years, who were infected with HIV-1 in 2013.

According to the World Health Organization (WHO) TDR surveillance threshold,¹⁰ we enrolled in the study a total 152 of 214 treatment-naive youth who were newly diagnosed with HIV-1 in 2013 and who were from all 11 prefectures of Hebei Province (Table 1). All participants signed written informed consent forms. Ethical approval was obtained from the local Ethics Committee at Hebei Provincial Center for Disease Control and Prevention.

Table 1.

Demographic Characteristics of the Study Subjects and the Distribution of HIV-1-Transmitted Drug Resistance

	Subjects related to TDR
Characteristics	Subjects	Cases	%	χ ²	p
Gender
Male	139	8	5.8	F	0.233
Female	13	2	15.4
Marital status
Married/cohabitation	31	3	9.7	F	0.439
Single^a	121	7	5.8
Ethnicity
Han	148	10	6.8	F	1.000
Minority	4	0	0
First CD4 count (cells/μl)
≤200	61	6	9.8	1.800	0.339
201–500	76	3	3.9
>500	15	1	6.7
Transmission route
MSM	120	7	5.8	1.411	0.500
Heterosexual	30	3	10.0
IDU	2	0	0
Subtype
CRF01_AE	81	8	9.9	4.975	0.306
CRF07_BC	40	0	0
CRF08_BC	2	0	0
B	26	2	7.7
URF	3	0	0
Prefecture
Baoding	17	0	0	6.412	0.729
Cangzhou	21	2	9.5
Chengde	9	0	0
Handan	6	0	0
Hengshui	2	0	0
Langfang	7	0	0
Qinhuangdao	13	0	0
Shijiazhuang	32	3	9.4
Tangshan	35	4	11.4
Xingtai	3	1	33.3
Zhangjiakou	7	0	0

Single include unmarried, widowed, and divorced.

F, Fisher-exact test; IDU, intravenous drug use; MSM, men who have sex with men; TDR, transmitted drug resistance; URF, unique recombinant form.

Participants’ demographic data were obtained by face-to-face interviews using a standard questionnaire during collection of their blood samples. Within 6 h of collection, 500-μl plasma samples were separated from whole blood and were used to extract, amplify, and sequence HIV-1 RNA.

An HIV-1 partial pol gene (HXB2:2147–3462) was amplified, sequenced, edited, and assembled using an in-house method, as reported previously.¹¹ The HIV-1 subtype was determined using the neighbor-joining (NJ) phylogenetic tree analysis. Reference sequences (A-D, F-H, J, K, CRF01_AE, CRF08_BC, and CRF07_BC) were obtained from an HIV database (www.hiv.lanl.gov/content/index). The NJ phylogenetic tree was constructed using MEGA 5.0 with 1,000 bootstrap replicates, based on the Kimura 2-parameter model. The mosaic structure of the novel recombinant form was analyzed using the online software programs jpHMM-HIV (http://jphmm.gobics.de/submission_hiv.html) and RIP 3.0 (www.hiv.lanl.gov/content/sequence/RIP/RIP.html).

Viral pol gene sequences were submitted to the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/pages/algs/HIVdb.html) and then compared with a gene sequence of a subtype B reference strain (HXB2). TDR mutations were evaluated according to the 2009 WHO TDR mutations list.¹² Levels of HIV-1 DR were analyzed using the Stanford DR algorithm. Statistical analysis was performed using IBM SPSS 21.0 (IBM Corp., Armonk, NY).

Demographic characteristics of 152 young people infected with HIV-1 are summarized in Table 1. Among these, 91.4% (139/152) were male and 79.6% (121/152) were unmarried. Han nationality accounted for 97.4% (148/152) of the population. Mean age was 21.9 (16–25) years and mean first CD4 cell counts were 270.8 (16–1149) cells/μl. A total of 98.7% (150/152) participants had a history of sexual exposure to HIV, including MSM (78.9%, 120/152) and heterosexuals (19.7%, 30/152). Another 1.3% (2/152) had a history of intravenous drug use (IDU).

In this study, phylogenetic analysis of 152 pol gene sequences (Fig. 1) indicated that the predominant subtype was CRF01_AE, accounting for 53.3% (81/152), followed by CRF07_BC (26.3%, 40/152), subtype B (17.1%, 26/152), unique recombinant forms (URFs; 2.0%, 3/152), and CRF08_BC (1.3%, 2/152). Mosaic structure analysis identified that the recombinant pattern of all three URFs was CRF01_AE/BC.

FIG. 1.

The phylogenetic tree analysis based on HIV-1 partial pol gene sequences from 152 newly diagnosed HIV-1 infected youths without the treatment in Hebei. The neighbor-joining phylogenetic tree was constructed with our sequences and the reference gene sequences of subtype A-D, F-H, J, K, O, CRF01_AE, CRF07_BC, and CRF08_BC obtained from the HIV database (www.hiv.lanl.gov/content/index) using MEGA 5.0 with 1,000 bootstrap replicates, based on Kimura 2-parameter Model. Bootstrap values ≥60% are shown in the tree. The scale length indicates 2% nucleotide sequence divergence. Black triangle denotes URFs. Black dot denotes the reference sequences. Black square and black triangle denote TDR mutations and TDR-associated mutations. Within the reference sequence ID, LN, YN, HN, XJ, BJ, and GX(gx) denote Liaoning, Yunnan, Henan, Xinjiang, Beijing and Guangxi, respectively. C, cluster; TDR, transmitted drug resistance; URFs, unique recombinant forms.

Table 2 reveals that 13 participants harbored DR to protease inhibitors (PIs; 7 cases) and nonnucleoside reverse transcriptase inhibitors (NNRTIs; 6 cases). Only one mutation was found in each of these 13 participants. M46L/I, Y181C, K101E, and G190E were included on the 2009 WHO TDR mutation list. The overall prevalence of TDR among participants was 6.6% (10/152), a moderate level (5.0%–15.0%) according to WHO Threshold Survey guidelines.¹³ This is higher than the prevalence of HIV TDR in 20 countries (3.7%)¹⁴ and in 19 provinces or cities (4.9%) of China.¹⁵ Furthermore, the prevalence of TDR after 10 years of ART scale-up in Hebei (6.6%) is clearly higher than the 3.2% after 8 years in Guangxi Province¹⁶ and the 4.3% after 7 years in Dehong Prefecture of Yunnan Province¹⁷; however, this rate is lower than the 14.3% after 8 years of ART scale-up in Henan Province.¹⁸

Table 2.

Epidemiological Characteristics of Subjects Associated with HIV-1-Transmitted Drug Resistance

ID	Prefecture	Gender	Age	Ethnicity	Marital status	Transmission route	First CD4	Subtype	PIs	NNRTIs
12126	Cangzhou	Male	23	Han	Married/cohabitation	Heterosexual	37	B		Y181C
12163	Shijiazhuang	Male	23	Han	Unmarried	MSM	247	B		K101E
12238	Tangshan	Male	25	Han	Married/cohabitation	MSM	345	CRF07_BC		V108I
12249	Tangshan	Male	23	Han	Unmarried	MSM	423	CRF01_AE		G190E
12267	Shijiazhuang	Male	19	Han	Unmarried	MSM	321	CRF01_AE	M46I
12325	Xingtai	Male	25	Han	Married/cohabitation	MSM	124	CRF01_AE	M46L
12460	Chengde	Male	19	Han	Unmarried	MSM	97	CRF07_BC		F227FL
12482	Cangzhou	Female	19	Han	Unmarried	Heterosexual	110	CRF01_AE	M46I
12634	Shijiazhuang	Female	25	Han	Married/cohabitation	Heterosexual	190	CRF01_AE	M46I
12643	Cangzhou	Male	22	Han	Unmarried	MSM	185	B		V108I
12718	Tangshan	Male	18	Han	Unmarried	MSM	775	CRF01_AE	M46I
12736	Tangshan	Male	20	Han	Unmarried	MSM	181	CRF01_AE	M46I
12740	Tangshan	Male	24	Han	Unmarried	MSM	36	CRF01_AE	M46I

Underlined bold mutations are not included in the 2009 World Health Organization TDR mutation list.

NNRTIs, nonnucleoside reverse transcriptase inhibitors; PIs, protease inhibitors.

As shown in Figure 1, those sequences containing TDR mutations (black squares) and TDR-associated mutations (black triangles) formed eight different clusters within CRF01_AE, subtype B, and CRF07_BC. Within CRF01_AE, six clusters containing TDR were found, including cluster 2 in the heterosexual group, cluster 3 in the MSM group, and another four clusters in the MSM and heterosexual groups. In particular, cluster 2 was associated with Guangxi (GX), and clusters 3 and 6 were closely associated with Liaoning (LN). Within subtype B, one DR transmission cluster (cluster 7) containing eight MSM sequences and three heterosexual sequences were identified, being related to Henan (HN) and Beijing (BJ). Within CRF07_BC, cluster 8 included sequences from the MSM (eight cases), IDU (one case), and heterosexual (two cases) groups. Those sequences harboring TDR-associated mutations were related to provinces with high HIV prevalence in China, such as Xinjiang, Yunnan, and Guangxi. The above facts show that DR is spreading rather rapidly among the population through sexual contact.

The main mutation associated with TDR to PIs was M46L/I, which had a prevalence of 4.6% (7/152). Three TDR mutations were identified in the NNRTI coding region, including Y181C (0.7%, 1/152), K101E (0.7%, 1/152), and G190E (0.7%, 1/152). The prevalence of TDR to NNRTIs was 2.0% (3/152). The prevalence of TDR to PIs and NNRTIs was at low levels (<5.0%). No TDR to NRTIs was found in this study.

As indicated in Tables 1 and 2, TDR mutations were mainly found in Shijiazhuang (9.4%, 3/32), Tangshan (11.4%, 4/35), Xingtai (33.3%, 1/3), and Cangzhou (9.5%, 2/21) prefectures; no TDR was observed in other prefectures. TDR mutations were observed in CRF01_AE (M46I/L and G190E) and subtype B (Y181C and K101E) only. The prevalence of TDR in CRF01_AE and subtype B was 9.9% (8/81) and 7.7% (2/26), respectively. All TDR mutations were found only in young people infected with HIV-1 through sexual activity, and the prevalence of TDR among heterosexuals (10.0%, 3/30) was higher than that among MSM (5.8%, 7/120). Although no TDR mutations were found for some demographic characteristics, the distribution of TDR by sex, age, first CD4 count, transmission route, marital status, ethnicity, subtype, and prefecture showed no significant differences (Table 1).

According to the Stanford DR algorithm, M46I/L can confer low-level resistance to nelfinavir (NFV). NFV has never been used in Hebei. We can infer that M46I/L may be derived from HIV-1-resistant strains in other provinces or foreign countries rather than PI selection in Hebei. Y181C is responsible for significant resistance to efavirenz (EFV), etravirine (ETR), rilpivirine (RPV), and nevirapine (NVP). K101E causes intermediate resistance to NVP and low-level resistance to EFV, ETR, and RPV. G190E results in high-level resistance to NVP and EFV and intermediate resistance to RPV and ETR.

In addition, both V108I and F227FL, which were not included on the 2009 WHO TDR surveillance list,¹² are nonpolymorphic TDR-associated mutations. V108I is an accessory mutation identified in two MSM (from Tangshan and Cangzhou) harboring CRF07_BC and subtype B (Table 2), respectively, and can result in low-level resistance to NVP and EFV. F227FL, detected in one MSM (from Chengde) with CRF07_BC (Table 2), is closely associated with intermediate resistance to NVP and low-level resistance to EFV.

Of the above drugs, EFV, ETR, and NVP are first-line antiretroviral drugs that have been included in ART regimens since the launching of ART in 2003. This suggests that TDR mutations occur owing to selection for antiretroviral drugs.

Currently, sexual exposure has become the predominant route for the spread of HIV-1 in Hebei. Moreover, CRF01_AE, subtype B, and CRF07_BC are the main subtypes circulating in the province. This study is the first investigation of TDR among newly diagnosed, treatment-naive young people aged 16 to 25 years, infected with HIV-1 in Hebei, and reveals a moderate prevalence of TDR (6.6%) after 10 years of ART in the province. The circulation of TDR HIV-1 strains among youth who are sexually active will cause rapid development of HIV-1 genetic diversity and reduce the effort to improve therapy outcomes. Our findings suggest that it is important to continually monitor HIV-1 TDR among treatment-naive young people who have been newly diagnosed with HIV-1 and who are sexually active. Medical interventions as well as interventions for high-risk sexual behavior are needed to help manage the HIV-1 epidemic in Hebei, China.

Footnotes

Acknowledgments

The authors thank the staff of local Centers for Disease Control and Prevention in Hebei Province for their assistance, especially with the fieldwork. This study was implemented with the support of the Natural Science Fund of Hebei Province (H2016303006). All nucleotide sequences reported in this study have been submitted to GenBank with accession numbers KX198563, KX198583, KX198569, KX198571, KX198581, and KX791498-KX791637.

Authors’ Contributions

The study was conceived and designed by X.L. and X.K. X.L. and Y.L. carried out the study and analyzed the experimental data. X.L. drafted the article. Y.L. edited and assembled sequences. Y.L., J.L., Z.C., and S.C. contributed to the acquisition of epidemiological data. All authors read and approved the final article.

Author Disclosure Statement

No competing financial interests exist.

References

Nwobegahay

, Bessong

, Masebe

, et al.: Prevalence of antiretroviral drug resistance mutations and HIV-I subtypes among newly-diagnosed drug-naïve persons visiting a voluntary testing and counselling centre in northeastern South Africa. J Health Popul Nutr, 2011; 29:303–309.

Hemelaar

: The origin and diversity of the HIV-1 pandemic. Trends Mol Med, 2012; 18:182–192.

Karlsson

, Björkman

, Bratt

, et al.: Low prevalence of transmitted drug resistance in patients newly diagnosed with HIV-1 infection in Sweden 2003–2010. PLoS One, 2012; 7:e33484.

Cortez

, Maldarelli

: Clinical management of HIV drug resistance. Viruses, 2011; 3:347–378.

Liu

, Liu

, Dang

, Liu

, Zhou

, Wang

: Analysis on the HIV/AIDS epidemic in Shijiazhuang. Modern Prevent Med, 2010; 37:3153–3154.

Chen

, Zhao

, et al.: Eighteen-year follow-up report of the surveillance and prevention of an HIV/AIDS outbreak amongst plasma donors in Hebei Province, China. BMC Infect Dis, 2015; 15:316.

, Kang

, Chen

, et al.: HIV-1 genetic diversity and transmitted drug resistance among recently infected individuals at men who have sex with men sentinel surveillance points in Hebei Province, China. AIDS Res Hum Retroviruse, 2015; 31:1038–1045.

Chen

, Ma

, Lu

, et al.: Effect of highly active anti-retroviral therapy on reducing HIV/AIDS related death in Hebei, 1989–2013. Zhonghua Liu Xing Bing Xue Za Zhi, 2015; 36:460–464.

, Kang

, Zhao

, et al.: Drug resistance mutations and their influencing factors in the patients infected with HIV-1 through different routes after antiretroviral therapy. Med J Chin PLA, 2015; 40:591–594.

10.

Bennett

, Myatt

, Bertagnolio

, Sutherland

, Gilks

: Recommendations for surveillance of transmitted HIV drug resistance in countries scaling up antiretroviral treatment. Antivir Ther, 2008; 13(Suppl 2):25–36.

11.

, Zhong

, Guo

, et al.: Prevalence and mutation patterns of HIV drug resistance from 2010 to 2011 among ART-failure individuals in the Yunnan Province, China. PLoS One, 2013; 8:e72630.

12.

Bennett

, Camacho

, Otelea

, et al.: Drug resistance mutations for surveillance of transmitted HIV-1 drugresistance: 2009 update. PLoS One, 2009; 4:e4724.

13.

Myatt

, Bennett

: A novel sequential sampling technique for the surveillance of transmitted HIV drug resistance by crosssectional survey for use in low resource settings. Antivir Ther, 2008; 13(Suppl 2):37–48.

14.

World Health Organization: HIV drug resistance fact sheet. J Assoc Nurses AIDS Care, 2001; 12:95–96.

15.

Yang

, Xing

, Niu

, et al.: The emergence of HIV-1 primary drug resistance genotypes among treatment-naïve men who have sex with men in high-prevalence areas in China. Arch Viro, 2013; 158:839–844.

16.

, Liang

, Harrison

, et al.: Prevalence of transmitted HIV-1 drug resistance remains low in Guangxi, China, eight years after scale-up of highly-active antiretroviral therapy. Intervirology, 2014; 57:270–276.

17.

Chen

, Ma

, Duan

, et al.: Genetic diversity and drug resistance among newly diagnosed and antiretroviral treatment-naive HIV-infected individuals in western Yunnan: A hot area of viral recombination in China. BMC Infect Dis, 2012; 12:382.

18.

, Sun

, Zeng

, et al.: Relatively high prevalence of drug resistance among antiretroviral-naive patients from Henan, Central China. AIDS Res Hum Retroviruses, 2014; 30:160–164.