This study is an effort to establish an HIV-1 resistance-monitoring system in Puerto Rico and a continuation of the articles published in 2002, 2008, and 2010 that examined the prevalence of HIV-1 mutations and antiretroviral resistance in the island from 2000 to 2005 [15
]. As highlighted in our previous reports, statistically significant differences between genders were observed for both antiretroviral resistance levels and mutation incidences. It is of interest to note that some of the gender differences detected in this study were also observed in previous years. In the case of antiretroviral drug resistance, gender differences were recorded for nevirapine in 2004 and 2007. The statistically significant results for the reverse transcriptase mutation K70R have been noted for 2003 and 2006, while gender differences for K103N were observed in 2005 and 2007. Similarly, the protease resistance mutation L90M showed a divergent result between men and women in 2003, 2005, and 2006. Even though it is premature to draw conclusions based on the recurrence of these events, gender difference patterns may emerge once data from subsequent years are made available for analysis.
The results also provide a glimpse on the evolving nature of the HIV-1 viral strains circulating in Puerto Rico. As illustrated in , resistance to zalcitabine and lamivudine dominated the drug resistance landscape in 2006, following the trend observed in the previous years [16
]. Resistance to emtricitabine/lamivudine and nevirapine was more common in the intermediate years, followed by a codominance of nevirapine and efavirenz in the last year of the study. There is a dramatic difference in the range of antiretroviral drug resistance observed from 2006 to 2010. In the first year of the study, there was a difference of approximately 56 percentual points between the most abundant and the least common antiretroviral resistance levels, and the gap closed in the subsequent years until a difference of only 15 percentual points was obtained for 2010.
The reverse transcriptase mutation results indicate that M184V continues to be the most common mutation surveyed in our studies. This is consistent with reports based on large-scale HIV-1 genotypic analyses that identify M184V/I as the most prevalent nucleoside and nucleotide-analog-RT-inhibitor- (NRTI-) resistance mutations that overcome the effects of drugs like emtricitabine and lamivudine [18
]. M41L, the second most common mutation for 2006 and the third most common mutation for 2007, 2008, and 2010, is also a dominant NRTI-resistance mutation identified in the same studies. K103N, the third most prevalent mutation for 2006 and the second most common mutation from 2007 to 2010, is frequently observed among nonnucleoside-RT-inhibitor-(NNRTI-) resistance mutations that neutralize efavirenz and nevirapine [18
] and has been identified as a prevalent mutation in other retrospective studies [21
As in previous years, the most prevalent protease resistance mutation corresponds to L63, a position that shows a great variation of amino acid substitutions contributing to antiretroviral drug resistance [22
]. M36I, a mutation that allows faster in vitro
HIV-1 replication regardless of the presence of protease inhibitors, was the second most common mutation for 2006 [24
]. V77I, a variant that has been identified as an important emerging substitution in HIV epidemiology studies, was the second most common mutation from 2007 to 2010 [25
]. The third position was occupied by I13V in 2006, 2007, and 2009, while this place corresponded to I62V in 2008 and 2010. These mutations have been associated with poor virological response to antiretroviral treatments [27
The tables show a descending trend for most of the mutations, accompanied by an increase in the incidence of minority mutations. This observation is more evident in where mutations that did not appear on the top ten mutations in previous years started to dominate the results landscape by the end of the study period. For example, I15V, a mutation highly prevalent in Chinese HIV-1+
patients who are drug users [30
], appeared for the first time in 2007 with a prevalence of 9.5% (data not shown), doubled in 2008, and remained as one of the most common mutations for 2009 and 2010. This observation may be validated once the data corresponding to the subsequent years are included in the analysis. More information is needed before an ascending tendency of minority mutations can be established with certainty.
Although deaths of persons with HIV/AIDS reported to the national HIV/AIDS surveillance system and US 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 [31
]. Members of minority racial/ethnic groups have a higher propensity to discontinue antiretroviral (ARV) therapy, experience more virologic failure, and show elevated morbidity [32
]. In the particular case of Hispanics, this ethnic group has been identified as one of the socioeconomic disparities associated with suboptimal HIV care, including delayed HIV diagnosis and treatment, early discontinuation of therapy, higher ratios of progression of disease, and death [35
]. Gender disparities have also been observed in the United States, and minority women are increasingly disadvantaged [39
]. Poverty, low health literacy, lack of family support, limited access to transportation, patient-provider issues, the organizational infrastructure of the health care facility visited, and the perceived HIV stigma within their communities are some of the issues associated with the inequality [41
]. The aforementioned problems, along with the inclusion of gender-specific issues into management strategies for HIV-infected women, including preconception and reproductive counseling, should be taken into consideration while developing health plans for these patients [43
]. Research targeted towards the reduction of HIV-related disparities, including the understanding of HIV/AIDS prevalence in women and minority groups, the detection of emerging incidence trajectories in these groups, and obtaining more information on how HIV infection as a chronic disease affects these individuals and their communities remains a high priority for the National Institutes of Health Trans-NIH AIDS research initiatives [45
In the particular case of the race/ethnicity versus drug-resistance correlation, studies reporting HIV drug-resistance rates between races are contradictory since race is accounted for differences in some publications [46
], while others noticed no differences attributable to race [47
]. 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 [49
]. 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 [50
]. A recent analysis of the data collected in ten studies found that the risk for virologic failure among patients with similar variant loads was higher among black and Hispanic patients. This relationship persisted, even after adjustment for differences in adherence rates, suggesting that socioeconomic factors, differences in levels of drug or alcohol abuse, or perhaps race-specific polymorphisms in the cytochrome P450 system may play an important role in this difference [51
Gender differences in ARV treatment outcomes and drug-resistance mutations are also of interest. Several studies from developed countries have not identified gender as a predictor for primary drug resistance [52
], while others report that mutation prevalence is higher in males [46
]. In a recent study, females showed 2-fold odds of having virological failure compared with males at one year after genotype resistance testing, independent of race or a history of optimal treatment [55
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 [56
], and many female participants withdraw their consent prior to the conclusion of the study [58
]. Similar pharmacokinetics, treatment responses, and outcomes are recorded for men and women [60
], but females are more susceptible to ARV treatment delay [63
], higher drug exposure due to lower weights [65
], and physiological and metabolic differences affecting drug absorbance, toxicity, and retention [57
]. Malaise symptoms like rash, peripheral neuropathy, fatigue, weight loss, and feelings of vertigo/dizziness are frequently reported by women [67
]. The aforementioned reasons, along with the psychosocial factors affecting treatment compliance [67
], account for a higher rate of treatment changes and poor adherence among women living with HIV.
Current evidence demonstrates that HIV ARV drug-resistance in resource-limited sites has neither emerged nor been transmitted to the degree that had initially been feared. However, due to a lack of standardized methodologies, HIV ARV resistance data from resource-limited sites can be difficult to interpret and may not provide the programmatic evidence necessary for public health action [69
]. As predicted, the use of ARV drugs in resource-rich regions has exerted an increased evolutionary pressure on the virus, leading to a higher prevalence of antiretroviral drug resistant variants even among treatment-naïve individuals. In the particular case of the United States, a cohort study conducted in New York city found that the prevalence of overall transmitted resistance changed from 13.2% to 24.1% during the periods of 1995 to 1998 and 2003 to 2004 [70
], and other studies reported an overall prevalence of transmitted drug resistance between 5 and 20% [71
]. Even though no specific drug resistance data has been published for Puerto Rico, resistant rates may be similar between Puerto Rico and the continental United States due to the observed shared infection pattern maintained by the high level of travel between the two jurisdictions.
Our study is limited by the lack of demographic and behavioral data that would allow the performance of multivariate statistical analyses that could explain the driving forces behind the reported statistically significant differences. Likewise, clinically relevant comparisons between drug-naïve and drug-experienced patients cannot be performed because the patients included in the study did not disclose their clinical history. TruGene was designed to detect HIV-1 subtype B, and even though some reports indicate that it can perform well on diverse HIV-1 subtypes [73
], we do not know if the increase in prevalence of minority mutations is affected by the introduction on nonsubtype B variants in the studied population. Furthermore, the assay detects mutations in the coding regions of the RT and protease genes, but it was not designed to identify mutations in other regions of the viral genome that could contribute to ARV therapy resistance.
According to the manufacturer, the rules for the TruGene HIV-1 gentotyping assay are developed from the knowledge of a world-renowned panel of HIV experts that meet annually to review the latest HIV-1 clinical and research data. The resistance effects of mutations identified in the HIV-1 sample by the OpenGene DNA sequencing system are the culmination of a proprietary interpretative algorithm that considers published and nonpublished data, the recommendations of the annual expert review panel as well as alternative interpretative algorithms. Six TruGene guideline rules were released during the study period (version 10 in January of 2006, version 11 in September of 2006, version 12 in February of 2007, version 13 in November of 2007, version 14 in February of 2009, and version 15 in December of 2009) and present a major technical limitation. Since privacy issues impede the electronic storage of patient data, previously analyzed samples cannot be harmonized by being reanalyzed using the latest software version that may detect formerly unknown resistance patterns. Therefore, the periodic actualization of the software to reflect the current state of knowledge of mutation-resistance correlations can introduce artifacts that are beyond the control of the investigators. For example, shows a shift in antiretroviral drug resistance from lamivudine in 2006 to emtricitabine in 2007 to lamivudine/emtricitabine for the rest of the study. Since both drugs are associated with the same major mutations [18
], it is possible that the change in the mutation-resistance correlation is directly related to software updates made by the manufacturer. Therefore, it is recommended to emphasize the results reported for the actual mutation rates because the outcome will remain the same, even if the mutation's correlation to a specific drug changes in the future to accommodate the evolving nature of our understanding of the HIV antiretroviral drug resistance field.
Like many commercial screening platforms, TruGene is also affected by its inability to detect the presence of minority viral variants that can rapidly grow under drug selection pressure and can contribute to treatment failure [74
]. This outcome may change once new high-throughput sequencing methods with the ability to quantify resistant variants, providing both proportional and absolute numbers of sequencing reads with a mutation, are readily available for clinical use. For example, ultradeep sequencing, a promising emerging technology, has been used to interpret ARV resistance in clinical trials [78
], and the ability of clinical laboratories to perform the method was examined in an international collaborative study [80
]. On the other hand, before these new sensitive resistance technologies can be used to improve the clinical utility of viral genotyping in a cost-effective manner, important issues like a better definition of the level of sensitivity required to detect drug-resistant variants, the effects different variants have on treatment response, and the requirement for genotypic assays to provide information on resistance mutation linkage must be addressed by the scientific community [81
Aside from the aforementioned limitations, the fact that gender differences in HIV mutations and ARV drug resistance in Puerto Rican patients have been noted in the past ten years could point out possible differences in ARV treatment efficiency for this particular population. Our data could be of value for prospective cohort studies designed to study these differences in more detail, with the goal of establishing HIV mutation and resistance models tailored to the needs of Hispanics in general and Puerto Ricans in particular.