Several studies have shown that the use of commercial genotyping kits to detect HIV-1 drug resistance lack the ability to detect minor variants, mutations that are less than approximately 20% of the clinical sample, and newer techniques can determine resistance with much greater sensitivity. These new methods that detect fewer than 20% of resistance mutations are important tools in furthering the understanding of mutations that arise with the use of single-dose nevirapine and will ultimately provide valuable insight into the clinical impact of low levels of resistant virions. The results of this in vitro analysis demonstrate that naturally occurring nucleotide polymorphisms in the binding site for the allele-specific primer in the ASPCR technique can have significant impact on the accurate quantitation of drug resistance variants.
While the clinical implication of minor variant drug resistance is unclear, a recent study from Botswana has demonstrated that exposure to nevirapine during delivery impacts negatively on maternal outcome with HAART if the medications are started within 6 months postpartum (Lockman et al., 2007
). The fading of drug resistance mutations after nevirapine exposure may be responsible for the successful outcomes seen when patients delay initiation of NNRTI-containing HAART.
The studies from developing countries that use single-dose nevirapine are quite heterogeneous with respect to the techniques used and the frequency of nevirapine resistance mutations detected in different HIV-1 subtypes (Flys et al., 2005
; Johnson et al., 2005
; Lalonde et al., 2007
; Loubser et al., 2006
; Palmer et al., 2006a
). All of the studies demonstrate that the commercial techniques are suboptimal in their ability to detect minor variants, but no consensus emerges from the data regarding the degree of resistance and the duration of persistence of minor variant resistance after nevirapine exposure. It is possible that some of the heterogeneity in results could be attributed to the different assays used, and an overestimation of resistance may occur in some cases, as demonstrated in this in vitro study. Techniques such as the oligonucleotide ligation assay (OLA) or assays dependent on hybridization probes may be subject to similar problems as the ASPCR assay in that the polymorphism in the region being studied may lead to differential binding of oligonucleotides or probes impacting accurate quantitation as seen with the ASPCR primers. The ability of the new genotyping methods to accurately detect and quantify resistance needs to be closely evaluated so that results using the various methods involving different HIV-1 subtypes can be accurately compared.
The discrepancies in the results seen in this study follow a predictable pattern when a universal ASPCR primer set for a specific subtype and polymorphism specific primers are used. The data demonstrate that with polymorphisms in the template close to 3′ end of the allele specific primer, the accuracy of the results is significant impacted. This was clearly demonstrated when looking for the K103N mutation (AAA→AAC) at position 103 of reverse transcriptase, the most commonly seen mutation in clinical samples from Botswana in women who received nevirapine for PMTCT (Shapiro et al., 2006
The mechanism responsible for the different results when the different sets of primers are used on the same template likely reflects the natural difference in bond strength that occurs between nucleotides. As a result of the three hydrogen bonds between the G-C nucleotides compared to the two hydrogen bonds between the A-T nucleotides, the G-C pair is more stable. The impact of this increased bond strength is critical when it occurs at the 3′ end of the allele specific primer. In the case of the K103N mutation (ntAAC), the mutation specific primer that ends in a guanine (G) and binds to the cytosine (C) will bind more tightly than the thymine (T) in the wild-type primer as it binds to the A (adenine). In the setting of other polymorphisms in the primer binding site, the terminal G-C binding would act as an anchor for the mutation primer with relatively increased (ie. not as significant of a decrease) binding to the template compared to the wild-type allele specific primer and its A-T bond. Polymorphisms close to the 3′ end of the primer and multiple polymorphisms in the binding site create a scenario that makes the terminal bond more significant in the allele specific reactions. In essence, the crossing threshold (CT) of the wild-type reaction (ntAAA) is shifted further to the right (amplifies later) compared to the CT of the mutation primer (ntAAC). When applied to the corresponding standard curve, this change will be calculated as less total wild-type template present in a sample than truly exists, thereby falsely underreporting wild-type and overcalling the 103N (ntAAC) mutation.
Each group analyzed in our data showed vast improvements in the accuracy of the assay when polymorphism specific primers were used, with the exception of Group II, which had a single polymorphism at a site distant from the 3′ end of the primers. Additionally, the ordering of the groups generally followed the anticipated increasing error rate between the two methods. Overall, the proximity of the polymorphisms to the 3′ end and the number of polymorphisms demonstrate clear situations where the ASPCR method falters unless primer binding site polymorphism specific primers are used.
Our technique differs from the protocol developed by others using universal primers specific to samples of HIV-1 subtype C (Palmer et al., 2006a
). In this previous approach, a common reverse primer was used that did not overlie the site of interest and this allows for the quantitation of a total number of copies of amplicon in each sample. The three primers (wild-type and both 103N mutation primers) were each used on the samples and the results were calculated based on a percentage of each amplicon over the total copy number obtained by the reverse primer. No attempt was made to quantify the infrequent ntAAG (103K) polymorphism in their analysis. It was decided to eliminate the common primer from the calculation and substitute a fourth primer containing the ntAAG polymorphism at position 103 as this would enable us to detect the four possible nucleotides at the position of interest without the need to calculate total copy number by a separate reaction, and be able to detect AAG when it is actually present in our samples rather than inferring it. The calculated number of copies from all four primers served as the total copy number and calculated the % wild-type or mutant based on number of copies that each represented of this total.
The use of three specific primers and one common primer (for calculation of the total copies) rather than four specific primers would not negate the effect of the binding site polymorphisms on the results seen. The ntAAC at position 103, when polymorphisms exist in the binding site, would still result in more robust amplification by comparison to the ntAAA or ntAAT. Whether the total copies is calculated separately (using a common primer) or by summing the four different nucleotide amplifications, the effect is the same in that the numerator will be falsely increased in the case of ntAAC due to the strength of the terminal G-C anchor. The resulting calculation predicts a falsely elevated percentage of minor variants.
The in vitro design of this study allows for close scrutiny of the technique prior to its application to clinical samples. The 8 different groups of binding-site polymorphisms in this analysis reflect those seen in a limited sample set from Botswana. Importantly, the polymorphisms seen are reflective of the heterogeneous nature of this region of reverse transcriptase. The consensus sequences shown in this report only include the areas of polymorphism tested in our experiments. The HIV-1 subtypes found in Africa all have changes in this region that could impact the ability to correctly apply ASPCR. Further heterogeneity exists in the region of amino acid 104 in all subtypes and at position 105 in subtypes B and C (data not shown) that were not seen in our samples. These variations in the genome would be expected to cause greater discrepancy in amplification of the ASPCR primers as they reside close to the 3′ end of the primers, and would likely overcall minor variant drug resistance at position 103.
The limitations of this technique as it applies to clinical samples remains the fact that sequencing of the region of interest must occur prior to applying the ASPCR technique in order to determine which primer pair set must be used in the analysis. The bulk sequencing technique used to determine the primers will not detect minor polymorphisms in the binding site, and this will possibly mitigate the exact determination of the percentage of minor variants. However, the majority of the variants in the binding site will be detected and by using the appropriate primers and corresponding standard curves, it will allow for closer approximation of the true drug resistant population.
The clinical significance of minor drug resistant variants remains unknown but the determination of a threshold of drug resistance below which good clinical outcomes can be expected is a potential use of this research particularly as it relates to women who received single-dose nevirapine for PMTCT. The use of highly sensitive techniques must be accompanied by careful examination of the impact of polymorphisms on theses assays. This in vitro examination of the ASPCR assay confirms the ability of ASPCR technique to detect minor drug resistance variants, but specific matching primer sets should be applied to clinical samples to ensure template specificity as minor changes in the area of drug resistance mutations can impact the results dramatically.