Prevalence Of Drug Resistance And Associated Mutations In A Population Of HIV-1+ Puerto Ricans In 2005

Abstract

This is a continuation of our efforts to maintain a record of the evolution of HIV-1 infection in Puerto Rico by monitoring the expression levels of antiretroviral resistance-associated mutations. Samples from 2005 were analyzed (458: 270 males, 137 females, 51 anonymous), using the TRUGENE HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System. Results show that 60.1% of males and 50.2% of females had HIV-1 with resistance to at least one medication. The average number of HIV mutations in males was 6.27, while the average number of HIV mutations in females was 5.49. The highest levels of resistance were to Zalcitabine, Lamivudine, and Stavudine. The reverse transcriptase mutations with the highest frequency of expression were M184V, K103N and D67N. Protease mutations with the highest rate of expression were L63P, M36I and L90M. Significant differences between men and women were recorded in the levels of HIV-1 expressed mutations and resistance.

INTRODUCTION

The HIV infection in Puerto Rico is characterized by patients with a medium age of 35, mostly Hispanic (98.8%) males (77.7%) and differs from the characteristics of other countries, including the USA, because the major risk of infection in Puerto Rico is drug abuse (54.3%) (1, 2). Puerto Rico has one of the highest incidences of HIV-1/AIDS cases per 100,000 population in the USA. For 2005 the number of persons living with AIDS was 10,685 with 29,092 cumulative cases, 42% related to drug use, and a per capita rate of 26.4/100,000 population (3) while for 2007 the numbers had increase to 11,503 living with AIDS, 36,736 cumulative cases, 40% related to drug use, with a per capita rate of 21.5/100,000 (4).

The number of deaths attributed to HIV has noticeably decreased in regions of the world that have full access to antiretroviral drugs (5). Currently, HIV therapy typically includes a combination of reverse transcriptase (RT) inhibitors, protease inhibitors (PI), integrase inhibitors, and fusion or entry inhibitors that inhibit HIV at different stages of the viral life cycle (6). However, there is concern about the development and transmission of drug-resistant HIV strains since the combined genetic diversity of the virus and the high resistance mutation rate produce numerous resistant strains (7) that affect treatment efficiency leading to virologic failure and increased mortality (8). Therapy for persons infected with multidrug-resistant HIV can be complicated and create difficult patient management issues for clinicians (9). Furthermore, HIV-infected persons with resistant strains continue HIV risk behaviors and transmit resistant strains to newly infected persons (10).

HIV-genome sequence data has been collected and analyzed in Puerto Rico since 2000 for the RT and the protease genes (11, 12). The activity of HIV-1 RT is essential for viral replication and is specifically required for the conversion of single-stranded viral RNA into double-stranded viral DNA, which is later integrated into the host genomic DNA. For this reason, HIV-1 RT inhibitors are powerful inhibitors of HIV-1 replication and represent an important class of antiretroviral agents. The HIV-1 protease cleaves viral Gag and Gag-Pol polyproteins into structure and replication proteins that are necessary for the virus to become infectious and therefore, PI inhibitors are important for HIV therapy. In this study, we determined the prevalence of resistant genotypic mutations in a population of HIV-1+ Puerto Ricans tested for HIV-1 infection in 2005 and analyzed gender differences in mutation expression profiles to the two aforementioned genes. The highest mutation frequencies were M184V for RT inhibitor resistance and L63P for PI resistance. Significant differences were recorded for various viral mutations and drug resistance between men and women.

MATERIALS AND METHODS

Samples

HIV-1 genotyping results (458: 270 males, 137 females, 51 anonymous) for 2005 from the Immunoretrovirus Research Laboratory located at the Universidad Central del Caribe in Bayamón, Puerto Rico were analyzed. The sex of the patients was the only demographic data known. Patient clinical data were unknown.

RNA Isolation

HIV-1 viral RNA was extracted using the QIA-GEN QIAamp Viral RNA Mini Kit (QIAGEN, Valencia CA). Briefly, serum (140 μl) was added to buffer AVL containing Carrier RNA in a microcentrifuge tube. Following incubation at room temperature for 10 min, 560 μl of ethanol was added and mixed. An aliquot of 630 μl was added to the QIAamp spin column and centrifuged at 8,000 rpm for 1 min (2x). The QIAamp spin column was placed into a clean 2-ml collection tube. Then, 500 μl of Buffer AW1 was added and centrifuged at 8,000 rpm for 1 min, followed by 500 μl of Buffer AW2 and centrifuged at 14,000 rpm for 3 min. The QIAamp spin column was placed in a clean 1.5-ml microcentrifuge tube and 60 μl of Buffer AVE was added. The tube was incubated at room temperature for 1 min and centrifuged 8,000 rpm for 1 min. Viral RNA was stored at −80°C.

Mutational Analysis

Whole blood from HIV-1 infected patients was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Plasma was separated and stored at −80°C until isolation of RNA, which was followed by analysis using the TruGene HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System (Bayer Diagnostics, Tarrytown, NY).

Statistical Analysis

InStat 3 for Macintosh (GraphPad Software, Inc, La Jolla, CA) was used to perform the analysis. Chi-square test was performed. Statistical significance was set at P≤ 0.05.

RESULTS

General gender distribution of resistant mutations to antiretrovirals

The aim of this study was to assess the prevalence of genetic changes in either the HIV RT or protease genes in a cohort of patients from Puerto Rico. Serum samples from HIV patients from 2005 collected by the Immunoretrovirus Research Laboratory at UCC were analyzed using TruGene HIV-1 Genotyping Kit, an integrated system that includes target gene amplification and DNA sequencing chemistry that was developed for the detection of mutations in the HIV-1 protease and reverse transcriptase sequences. From a sample size of 458 patients, 60.1% of males and 50.2% of females had resistance to at least one medication. The average number of mutations in males was 6.27 while the average number of mutations in females was slightly less (5.49).

Levels of resistance to antiretrovirals as determined by TrueGene HIV-1 genotyping

As shown in Table I., the 2005 patients showed the highest levels of HIV-1 resistance to the following antiretrovirals: Zalcitabine (50.9%), Lamivudine (44.5%), and Stavudine (39.1%). In addition, there was a significant difference in resistance between males and females to Atazanavir (P=0.0103), Nelfinavir (P=0.0103), Saquinavir (P=0.0427), and Lopinavir + Ritonavir (P= 0.0103).

Table I.

HIV-1 Resistance for antiretrovirals as determine by TrueGene Numbers indicate the percentage of resistance against antiretrovirals expressed by HIV-1 as determined by the TrueGene system.

	% of resistance
Antiretroviral Drug	Men	Women	Total
Zalcitabine	54.6	50.7	50.9
Lamivudine	47.6	44.9	44.5
Stavudine	42.1	38.2	39.1
Abacavir	42.4	36.8	38.6
Atazanavir*	42.1	38.2	38.6
Nelfinavir*	44.6	30.9	38.0
Nevirapine	35.4	40.4	37.8
Efavirenz	35.4	40.4	37.8
Zidovudine	40.2	37.5	37.3
Delavirdine	33.2	38.2	35.8
Indinavir	36.5	28.7	31.7
Ritonavir	36.5	28.7	31.7
Saquinavir*	34.7	24.3	29.3
Tenofovir	29.2	25.7	26.4
Didanosine	28.0	25.7	26.2
Amprenavir	30.3	22.1	26.0
Lopinavir + Ritonavir*	29.2	16.9	23.8

^*indicates a significant difference between men and women.

RT resistance-associated mutations

Table II. compares the levels of RT resistance-associated specific mutations. Analysis of RT mutations for 2005 demonstrated that the highest degree of expression was for M184V (41.3%), K103N (25.3%), and D67N (20.5%). For women, P225H was among the top ten mutations (6.6%) while in males its expression was 1.9%. Significant differences between men and women for the expression of K103N (P=0.0456) and P225H (P=0.006) were recorded. Of interest is to note that all the aforementioned mutations have been recognized as major mutations associated with resistance to RT inhibitors (7).

Table II.

Most Prevalent HIV-1 RT Resistant Mutations Numbers express the percentage of expression of the most prevalent mutations as determined by the TrueGene system.

	% of expression
Mutation	Men	Women	Total
M184V	43.7	41.6	41.3
K103N*	21.1	30.7	25.3
D67N	21.9	20.4	20.5
M41L	22.2	19.0	20.3
V118I	18.1	14.6	16.6
T215Y	16.3	17.5	15.9
K70R	15.6	16.8	15.5
L210W	13.3	13.9	12.9
K219Q	12.6	12.4	11.6
L100I	8.1	5.8	7.4

^*indicates a significant difference between men and women.

PI resistance-associated mutations

As shown in Table III, the PI resistance-associated specific mutations with the highest degree of expression for 2005 were L63P (73.6%), M36I (26.2%), and L90M (21.0%). For women, N88D was among the top ten mutations (11.7%) while in males its expression was 9.3%. There was a significant difference between men and woman for L90M (P= 0.0337) and M46I (P= 0.0424), two major mutations associated with resistance to PI inhibitors (7).

Table III.

Most Prevalent HIV-1 PI Resistant Mutations Numbers express the percentage of expression of the most prevalent mutations as determined by the True-Gene system.

	% of expression
Mutation	Men	Women	Total
L63P	75.9	70.1	73.6
M36I	25.9	24.8	26.2
L90M*	25.9	16.1	21.0
L10I	22.6	20.4	20.5
A71V	23.0	16.1	19.4
M46I*	18.5	10.2	14.6
A71T	12.6	12.4	12.0
I54V	14.4	8.0	11.4
V82A	12.2	10.2	10.7
D30N	10.7	10.2	10.3

^*indicates significant difference between men and women.

DISCUSSION

This study is an effort to establish a HIV-1 resistance-monitoring system in Puerto Rico and a continuation of reports published in 2002 and 2008 that examined the prevalence of HIV-1 resistant mutations in the island from 2000 to 2004 (11, 12). Statistically significant differences were observed in mutation incidence and resistance between genders. In the case of the protease mutations, a statistically significant difference for L90M has been noticed since 2003 and the statistically significant difference observed for M46I in 2003 reappeared in 2005. K103N was the only statistically significant mutation in the RT gene obtained in the 2005 data. The statistical significance for Saquinavir noticed in 2004 remained in 2005 while the statistically significant difference for Nelfinavir noticed in 2003 reappeared in 2005. On the other hand, new statistically significance differences were observed for Atazanavir and Lopinavir + Ritonavir. These differences, although outside of the scope of the current study, deserve further attention. Their implications may be more evident once subsequent annual data are added to the analyses.

Although deaths of persons with HIV/AIDS reported to the national HIV/AIDS surveillance system and U.S. Vital Statistics have followed similar patterns across most demographic and behavioral strata, including gender, age, geographic distribution, and race/ethnicity; substantial variation exists in the percentages of decline among different subgroups (13). Even though no racial/ethnic or gender disparities have been observed in antiretroval (ARV) therapy receipt, minority racial/ethnic groups were faster to discontinue ARV therapy and experience virologic failure (14). Therefore, more research targeted towards the understanding of HIV/AIDS prevalence in women and minority groups is needed to detect emerging incidence trajectories in these groups and to determine how HIV infection as a chronic disease affects these individuals and their communities. In the particular case of the race/ethnicity vs. drug-resistance correlation, studies reporting HIV drug-resistance rates between races are contradictory since race is accounted for differences in some publications (10) while others noticed no differences attributable to race (15). In the case of ARV therapy tolerance, race has been correlated with alterations in metabolic and anthropometric measures where Latinos experienced the most unfavorable changes (16). Minorities are also at higher risks of experiencing specific adverse events but not in the overall adverse event rates, all-cause mortality, or rates of toxicity-related treatment discontinuations (17).

Gender differences in ARV treatment outcomes and drug-resistance mutations are also of interest. Treatments for HIV-infected women are usually based on efficacy and tolerability studies conducted in men since women are typically underrepresented in ARV treatment clinical trials (18). Pharmacokinetic data suggest similar response to treatment and similar outcomes in men and women but females are more susceptible to: (i) ARV treatment delay (19), (ii) experience more side effects (20), and (iii) higher drug exposure due to physiological and metabolic differences affecting drug absorbance, toxicity and retention (18). Consequently, women may experience more treatment changes than men and are more likely to poor treatment adherence due to physiological and psychosocial factors affecting treatment compliance (21).

This report contains the 10 most common mutations in the RT and the protease genes as well as their current ARV drug resistance interpretations. We observed statistically significant differences between males and females for several mutations. The K103N RT mutation was more common in women while the protease mutations L90M and M46I were more prevalent in men. Our results show that 60.1% of males and 50.2% of females tested in 2005 had HIV-1 with resistance to at least one medication. The average number of HIV mutations in males was 6.27, while the average number of HIV mutations in females was 5.49. Several cohort studies have not identified gender as a predictor for primary drug resistance (22) while others report that mutation prevalence is higher in males (10). More research is needed to establish correlations between HIV mutations, drug resistance and gender in predominantly Hispanic populations since people of Hispanic origin are underrepresented in many mutation prevalence reports.

HIV drug resistance is becoming a growing problem around the globe and monitoring must play a part in the surveillance and control of the epidemic worldwide (23). However, it has been difficult to compare national, regional, and local estimates of transmitted resistance because of differences in study design and because estimates have been based on different lists of resistance mutations (24). In one of the largest HIV drug resistance surveys ever performed in the USA, the estimated prevalence of HIV drug resistance was 76% with 48% of infected individuals harboring multidrug-resistant strains (25). Even though no specific data has been published for 2005, resistant rates may be similar between Puerto Rico and the USA due to the observed shared infection pattern maintained by the high level of travel between the 2 countries (26).

Our study is limited by the lack of demographic, clinical and behavioral data that would allow the performance of multivariate statistical analyses that could explain the driving forces behind the reported statistically significant differences. Furthermore, the assay can detect any mutation in the coding regions of the RT and protease genes but does not detect mutations in other regions of the viral genome that could contribute to ARV therapy resistance. In addition, software upgrades in the TRUGENE system present a technical limitation since the software is periodically updated to reflect the current state of knowledge of mutation – resistance correlations. Since privacy issues impede the electronic storage of patient data, previously analyzed data cannot be reanalyzed to detect formerly unknown resistance patterns. Commercial screening platforms are also affected by the presence of minority viral variants in patients that can rapidly grow under drug selection pressure and can contribute to treatment failure (27). However, the current techniques available to detect their presence are difficult to manage and are not likely to become part of routine clinical care in the near future (28). Until better technologies are available, it is standard practice to monitor HIV drug resistance using either genotypic or phenotypic resistance assays. The use of resistance assays can instruct drug selection, help in patient management, and improve therapy outcomes (29). The vast majority of adherent patients without resistance on standard resistance testing become virologically suppressed (28).

The fact that there are differences between sexes in HIV mutations in Puerto Rican patients could point out possible differences in ARV treatment efficiency. Our data could serve as baseline for prospective cohort studies where scientists, patients and physicians collaborate to study these differences in more detail with the goal of establishing HIV mutation and resistance models tailored to the needs of the Puerto Rican population.