Surveillance of Transmitted HIV Type 1 Drug Resistance in Newly Diagnosed HIV Type 1-Infected Patients in Shandong Province,China

Abstract

A survey to measure transmitted HIV-1 drug resistance (DR) was conducted in 2006 following the World Health Organization threshold survey protocol. Dried blood spots (DBS) were prepared from 53 newly HIV-1-diagnosed patients. Protease and reverse transcriptase (RT) gene regions were sequenced using a broadly sensitive genotyping assay and analyses to identify DR mutations and determine phylogeny of the HIV-1 strains were conducted. Forty-six of the 47 successfully genotyped DBS had no transmitted DR mutations; one had an NNRTI mutation (K101E) in the RT region. Phylogenetic analyses revealed 21 (44.7%) were CRF01_AE, 9 (19.1%) B, 6 (12.8%) CRF07_BC, 3 (6.4%) each of CRF08_BC and C, and 2 (4.3%) B/C unique recombinant forms (URF). The remaining three were one each of A/B, A/C, and unclassifiable. Our analyses indicate that the prevalence of transmitted DR in this population is low and the HIV-1 epidemic in the area was characterized by multiple subtypes and recombinant forms.

C hina, the most populous country in the world, is facing a significant public health threat from the HIV-1 epidemic as the virus is spreading from relatively localized high-risk groups to the general population. Since the first AIDS patient was identified in 1985, it is estimated that 700,000 people were living with HIV/AIDS at the end of 2007.¹ To curb the HIV/AIDS epidemic, the Chinese government launched the “Four Frees and One Care” policy in 2003.² By the end of 2007, 1190 counties were providing antiretroviral therapy (ART) to eligible HIV/AIDS patients, and there were 31,849 individuals on ART.¹

Shandong, located at the central eastern part of China, is a relatively developed coastal province with a population of more than 91 million people, making it the second most populated province in China. Since the first case of HIV-1 infection was reported in 1992,³ the HIV epidemic has spread to high-risk groups, including paid blood donors, commercial sex workers (CSW), men who have sex with men (MSM), and intravenous drug users (IDU). By the end of 2008, 1564 HIV/AIDS cases had been reported.³ With the rapid increase of HIV cases, Shandong, as a pilot province, developed a free scaling-up ART program in 2003. By the end of 2008, 310 patients had received free antiretrovirals (ARV) with standardized first-line regimens recommended by the World Health Organization (WHO),³ which include four nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), lamivudine (3TC), stavudine (D4T), didanosine (DDI), and zidovudine (ZDV), and two nonnucleoside reverse transcriptase inhibitors (NNRTIs), nevirapine (NVP) and efavirenz (EFV).

Because of the high replication and mutation rates of HIV viruses and life-long treatment requirement for ART patients, it has been widely feared that the rapid scaling-up of ART could lead to the emergence of drug resistance (DR), which slows efforts at ART expansion and jeopardizes the prevention and control of HIV infections. In 2003, WHO developed a global strategy for HIVDR prevention, surveillance, and monitoring, including the HIVDR threshold survey (HIVDR-TS).⁴

In 2006, 4 years after implementing the free ART scaling-up program in Shandong Province, we conducted this survey to estimate the prevalence of transmitted HIVDR in recently HIV-1-diagnosed patients. To increase the possibility that the specimens were collected from recently HIV-infected patients, we enrolled the individuals in the survey only if they were serologically tested HIV-1 positive for the first time and under 25 years of age. After receiving written consent, a questionnaire was administered to each participant to collect demographic, epidemiological, and clinical data. Between June and December 2006, we consecutively collected 53 blood specimens from qualified individuals at volunteering counseling and testing (VCT) sites in Shandong using the binomial sequential sampling strategy.⁵

Within 24 h of blood draw, dried blood spots (DBS) were prepared with Whatman 903 filter paper (Whatman Inc., Florham Park, NJ) by spotting 100 μl of the blood onto each of the five preprinted circles. After drying overnight at ambient temperature, the DBS were packaged⁶ and stored in a –80°C freezer until they were sent by express mail to the Centers for Disease Control and Prevention (CDC), Atlanta, GA, for analysis. Two DBS spots from each patient specimen were cut out for total nucleic acid extraction using a modified NucliSens method.⁶ The HIV-1 pol gene corresponding to amino acids 13–99 of the protease and 1–251 of the reverse transcriptase (RT) regions were amplified using a broadly sensitive in-house genotyping assay.⁷ The nested polymerase chain reaction (PCR) products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA) and sequenced using an ABI3730 DNA Genetic Analyzer (ABI, Foster City, CA). The sequences were edited with the ChromasPro version 1.42 (Technelysium Pty Ltd, Australia). Mutations related to transmitted HIVDR in the protease and RT gene regions were interpreted using the Calibrated Population Resistance (CPR) tool in the Stanford University HIV Drug Resistance Database (http://cpr.stanford.edu/cpr/) and the WHO List of Mutations for Surveillance of Transmitted Drug Resistant HIV Strains.⁸ These newly obtained sequences, along with reference sequences from the HIV sequence database (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html), were aligned by the CLUSTAL W (version 1.74) multiple sequence alignment program included in the BioEdit program.⁹ The genetic distances between strains were computed by DNADIST and a phylogenetic tree was constructed after all gaps had been trimmed using the neighbor-joining method included in the MEGA 4.1 package.¹⁰

The 53 eligible participants included 29 females and 24 males between the ages of 17 and 24 years. All participants were diagnosed with HIV-1 infection at VCT sites in 12 cities, with the majority from Jinan (11 patients), Linyi (13 patients), and Weifang (8 patients). These patients belonged to eight ethnic groups, including 26 from Han, five each from Jingpo, Yi, and Dai, four each from Achang and Wa, two from Uygur, one from Lahu, and one with unknown status. Transmission routes based on analysis of the questionnaire data included heterosexual transmission (37/53 or 69.8%), homosexual transmission (8/53 or 15.1%), and intravenous drug use (6/53 or 11.3%); the remaining two (3.8%) participants had unknown HIV transmission routes.

According to the WHO HIVDR-TS protocol, a minimum of 47 qualified sequences obtained from consecutively collected HIV-positive specimens are needed to classify transmitted HIVDR into different levels. In this survey, we were able to consecutively genotype 47 of the 53 DBS specimens (88.7%). Resistance analysis of the RT data using the WHO⁸ and CPR criteria indicated that among the 47 specimens, only one had a mutation (K101E) associated with transmitted HIVDR to NNRTI. This mutation is known to cause intermediate resistance to NVP and delavirdine (DLV) and low-level resistance to EFV and etravirine (ETR).¹¹ With only one (the 17th) specimen having transmitted DR mutation (K101E) among the 47 consecutive specimens analyzed, genotyping analysis was stopped at specimen 44 by the computer program when the running total was less than the lower limit (Table 1). This revealed a low (<5%) prevalence of transmitted DR to all relevant ARV drugs and drug classes. Five specimens had borderline/suspicious mutations associated with transmitted HIVDR in RT when CPR criteria were used; three (6.4%) had mutations (V118I) potentially against NRTIs and two (4.3%) had mutations (V179D) potentially against NNRTIs.

Table 1.

Sampling and Classification Plan for Reverse Transcriptase Mutations

Sample number genotyped (SNG)	Lower limit (LL)	Running total of specimens with HIVDR (RT)	Upper limit (UL)	Sample number genotyped (SNG)	Lower limit (LL)	Running total of specimens with HIVDR (RT)	Upper limit (UL)
1	ND^a	__0___	ND	25	ND	__1___	6
2	ND	__0___	ND	26	ND	__1___	6
3	ND	__0___	ND	27	ND	__1___	6
4	ND	__0___	ND	28	ND	__1___	6
5	ND	__0___	ND	29	ND	__1___	6
6	ND	__0___	ND	30	ND	__1___	6
7	ND	__0___	ND	31	ND	__1___	6
8	ND	__0___	ND	32	ND	__1___	6
9	ND	__0___	ND	33	ND	__1___	6
10	ND	__0___	ND	34	1	__1___	6
11	ND	__0___	ND	35	1	__1___	7
12	ND	__0___	ND	36	1	__1___	7
13	ND	__0___	ND	37	1	__1___	7
14	ND	__0___	5	38	1	__1___	7
15	ND	__0___	5	39	1	__1___	7
16	ND	__0___	5	40	1	__1___	7
17	ND	__1___	5	41	1	__1___	7
18	ND	__1___	5	42	1	__1___	7
19	ND	__1___	5	43	1	__1___	7
20	ND	__1___	5	44	2	__1___	7
21	ND	__1___	5	45	2	______	7
22	ND	__1___	5	46	2	______	8
23	ND	__1___	5	47	2	______	8
24	ND	__1___	5	STOP	STOP	STOP	STOP

ND, no decision.

Analysis of the HIV protease gene in the 47 specimens using the CPR criteria indicated that one (2.1%) specimen had a borderline/suspicious mutation (L33F) associated with transmitted DR. Eight specimens also had nine unusual mutations that may represent rare polymorphisms or novel DR mutations: G17D, L19M, K70Q, P79S, T91V, I93V, and F99L (3). In this survey, no specimen with transmitted DR mutations to protease inhibitors according to the WHO criteria was detected.⁸ Thus, the genotyping was stopped at specimen number 34 by the computer program when the running total was less than the lower limit, indicating a low (<5%) prevalence of transmitted protease inhibitor resistance (Table 2).

Table 2.

Sampling and Classification Plan for Protease Mutations

Sample number genotyped (SNG)	Lower limit (LL)	Running total of specimens with HIVDR (RT)	Upper limit (UL)	Sample number genotyped (SNG)	Lower limit (LL)	Running total of specimens with HIVDR (RT)	Upper limit (UL)
1	ND^a	__0___	ND	25	ND	__0___	6
2	ND	__0___	ND	26	ND	__0___	6
3	ND	__0___	ND	27	ND	__0___	6
4	ND	__0___	ND	28	ND	__0___	6
5	ND	__0___	ND	29	ND	__0___	6
6	ND	__0___	ND	30	ND	__0___	6
7	ND	__0___	ND	31	ND	__0___	6
8	ND	__0___	ND	32	ND	__0___	6
9	ND	__0___	ND	33	ND	__0___	6
10	ND	__0___	ND	34	1	__0___	6
11	ND	__0___	ND	35	1	______	7
12	ND	__0___	ND	36	1	______	7
13	ND	__0___	ND	37	1	______	7
14	ND	__0___	5	38	1	______	7
15	ND	__0___	5	39	1	______	7
16	ND	__0___	5	40	1	______	7
17	ND	__0___	5	41	1	______	7
18	ND	__0___	5	42	1	______	7
19	ND	__0___	5	43	1	______	7
20	ND	__0___	5	44	2	______	7
21	ND	__0___	5	45	2	______	7
22	ND	__0___	5	46	2	______	8
23	ND	__0___	5	47	2	______	8
24	ND	__0___	5	STOP	STOP	STOP	STOP

ND, no decision.

Results of the phylogenetic analysis revealed that among 47 newly obtained sequences, the predominant subtype was CRF01_AE (21, 44.7%), followed by B (9, 19.1%), CRF07_BC (6, 12.8%), CRF08_BC (3, 6.4%), C (3, 6.4%), B/C unique recombinant forms (URFs) (2, 4.3%), A/B URF (1, 2.1%), A/C URF (1, 2.1%), and unclassifiable (1, 2.1%). Overall, five HIV-1 subtypes/CRFs were identified and 72.3% of the infections were caused by CRFs and URFs (Fig. 1). These results indicate that viral recombination occurs frequently in these populations and repeated HIV-1 infections are not uncommon.

FIG. 1.

Phylogenetic classification of the newly characterized HIV-1 group M representative sequences (CN.SN#) along with reference sequences from the HIV sequence database (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) in the pol region. The unrooted tree was constructed by the neighbor-joining method using consensus sequences of 990 nucleotides. Branch lengths are drawn to scale, with the bar indicating 0.05 nucleotide substitution per site. Numbers at the nodes indicate the percentage of bootstrap sampling (out of 1000) in which the cluster is supported (only values ≥70% are shown). The boldface sequences represent new sequences from the current study. GenBank accession numbers for the newly characterized sequences are GQ443552–GQ443598. CRF, circulating recombinant forms; URF, unique recombinant forms; UC, unclassifiable.

The current survey for transmitted HIVDR in recently HIV-1-infected individuals in Shandong Province, China indicated that transmitted HIVDR to both RT and protease inhibitors was occurring at a low rate (<5%) in 2006 and that current ARV regimens are likely to remain effective when populations currently becoming infected in this region become eligible for therapy. Several reasons may possibly be responsible for this low HIVDR prevalence. First, almost all patients during the first 3 years of the ART program were paid blood donors, who might have transmitted HIV to their close contacts due to conservative sexual practice. Second, a high adherence to ART was practiced by patients.¹² Third, clinical data, including immunological and DR monitoring, from treated patients also showed complete viral suppression and improved immune status for almost all the patients.¹²

Our data showed that only one patient had an HIV strain with a transmitted DR mutation, K101E, which has been associated with intermediate resistance to NVP and DLV and low-level resistance to EFV and ETR.¹¹ Nevirapine is the first and most commonly used NNRTI in first-line regimens in Shandong. Together with EFV, they are the only NNRTIs that are accessible to patients in Shandong. Therefore, the occurrence of K101E may have a significant impact on the efficacy of first-line ARV regimens. Plans are needed to monitor the development of resistant viral strains in patients under ART. Although there is no evidence suggesting that the secondary DR mutations and unusual mutations detected in the study had a direct effect on ARV efficacy, some of them may have the potential to influence treatment outcomes.

The V118I in the RT region occurs in ∼2% of untreated persons, increases in frequency in persons receiving multiple NRTIs,^11,13 and can cause low-level resistance to 3TC and possibly to other NRTIs when present with E44A/D and/or one or more thymidine analog mutations (TAMs).¹³ The V179D/E can also cause low-level reductions in susceptibility to NNRTIs and occurs in about 1% of untreated persons.^11,13 A study showed that the combination of K103R and V179D reduces the susceptibility to NVP, DLV, and EFV by about 15-fold.¹³ Our study demonstrated that the frequency of V118I and V179D in the participants was 6.4% and 4.3%, respectively; these are much higher than expected in treatment-naive patients.¹³ We also detected minor mutations to PI. L33F is selected by fosamprenavir, darunavir, LPV, atazanavir, and tipranavir (TPV), and contributes slightly to resistance to these drugs.¹³

These mutations may influence the efficacy of ARV in HIV-1 strains that circulate in Shandong. Overall, the number of patients under ART is still low in Shandong; thus the chance to transmit DR strains might be minimal. However, as ART scaling up continues, the transmitted DR may become a public health threat. Therefore, monitoring DR development in treated patients and transmitted DR in recently infected patients are important to ensure that the current ART strategies remain effective.

One limitation of the survey was that many of the newly diagnosed HIV-infected patients were so called imported wives (IWs) who previously resided outside of Shandong and migrated to Shandong after marrying local residents. They might have been infected with HIV-1 from other areas, potentially making this survey not a full representation of the recently infected population in Shandong Province. This was probably responsible for the fact that the strains circulating in recently HIV-1-infected individuals participated in the survey were substantially different from those previously reported.¹⁴

In summary, this is the first transmitted HIVDR-TS conducted in China using DBS as a sample collection methodology. The survey indicated that transmitted DR in the newly diagnosed HIV-1-infected population is low. Our current survey and surveys reported before demonstrated that DBS is a valuable alternative for HIVDR genotyping in resource-limited settings.^15,16 Because of the genetic characteristics of HIV-1 viruses and the life-long treatment for HIV-infected patients, it is imperative to continue to conduct HIVDR surveillance in HIV epidemic areas in which ART coverage is growing.

Sequence Data

The sequences obtained in this survey were deposited in GenBank under accession numbers GQ443552–GQ443598.

Footnotes

Acknowledgments

Dr. Jing Zhang is a recipient of the International Emerging Infectious Diseases (EID) Laboratory Fellowship Program sponsored by Association of public Health Laboratories (APHL) and CDC. J. Zhang and D. Kang contributed equally to this article.

Disclosure Statement

No competing financial interests exist.

References

State Council AIDS Working Committee Office UN Theme Group on AIDS in China: A Joint Assessment of HIV/AIDS Prevention, Treatment and Care in China (2007). 12-1-2007.

State Council AIDS Working Committee Office UN Theme Group on AIDS in China: A joint Assessment of HIV/AIDS Prevention, treatment and Care in China (2004). 12-1-2004.

Shandong Provincial Health Department: HIV/AIDS Epidemic Report of Shandong (2008), 2008.

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.

Myatt

, Bennett

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

McNulty

, Jennings

, Bennett

et al. Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing. J Clin Microbiol, 2007; 45,2:517–521.

McNulty

, Diallo

, Zhang

et al. Development and application of a broadly-sensitive genotyping assay for surveillance of HIV-1 drug resistance in PEPFAR countries. Antivir Ther, 2008; 13,Suppl 3:A117.

Bennett

, Camacho

, Otelea

et al. Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PLoS One, 2009; 4,3:e4724Epub March 6, 2009.

Hall

. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser, 1999; 41:95–98.

10.

Tamura

, Dudley

, Nei

, Kumar

. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol, 2007; 24,8:1596–1599.

11.

Lazzarin

, Campbell

, Clotet

et al. Efficacy and safety of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-2: 24-week results from a randomised, double-blind, placebo-controlled trial. Lancet, 2007; 370:39–48.

12.

Zhang

, Lin

et al. Study on the effect of treatment and the status of drug resistance among HIV/AIDS patients treated with highly active antiretroviral therapy in Shandong province. Zhonghua Liu Xing Bing Xue Za Zhi, 2007; 28,11:1108–1111.

13.

Stanford Drug Resistance Database, 2008. http://hivdb.stanford.edu.

14.

, Li

, Zhang

et al. Study on the molecular epidemiology of people infected with human immunodeficiency virus-1 in Shandong province. Zhonghua Liu Xing Bing Xue Za Zhi, 2005; 26,2:124–127.

15.

Somi

, Kibuka

, Diallo

et al. Surveillance of transmitted HIV drug resistance among women attending antenatal clinics in Dar es Salaam, Tanzania. Antivir Ther, 2008; 13,Suppl 2:77–82.

16.

Kamoto

, Aberle-Grasse

. Surveillance of transmitted HIV drug resistance with the World Health Organization threshold survey method in Lilongwe, Malawi. Antivir Ther, 2008; 13,Suppl 2:83–87.