High levels of transmitted drug resistance persisted in 13 of 14 subjects. In only one person did a transmitted DR K103N variant become completely replaced by a WT virus such that it was not detectable by conventional population sequencing nearly 3 years after infection. Among all 14 subjects, the first detection of a mixture of WT and DR virus by population sequencing occurred at a mean of 96 weeks (1.8 years) after the EDI. Given the very low rate of resistance replacement, it was not possible to measure directly the time to complete loss of detectable drug resistance by bulk sequencing; rather, we imputed the time to this event using the lower 95% CIs (Fig. ). Despite a conservative estimate of a median time to complete replacement of 4.1 years, the transmitted resistant variant is archived for life, even when below the threshold of detection (34
). A less conservative estimate allows the lifelong persistence of detectable transmitted drug resistance in many people. The rate of replacement of the resistant mutant is likely to vary directly with the fitness cost associated with the resistance mutation and perhaps other host selective pressures not yet recognized. Although a significant in vitro fitness impairment has been observed for several of the major PI resistance mutations relative to WT virus (23
), we did not observe an increased rate of replacement of PI resistance mutations compared to that observed for NNRTI resistance mutations. Overall, replacement of resistance is gradual and usually incomplete, resulting in the persistence of mixtures of WT and resistant variants in plasma HIV RNA for years following initial infection.
The number of years after initial HIV infection during which detection of transmitted drug resistance is routinely feasible has not yet been defined, but patients identified many years, perhaps more than 10 years, after their EDI may not be properly identified as having been initially infected with DR virus (38
). The prevalence of transmitted DR HIV measured within the first year following HIV infection is between 20% and 23% in some North American populations and between 10% and 14% in some European and Canadian populations (12
) More recent estimates suggest that the prevalence of transmitted resistance may be declining in both North America and Europe (S. Little, S. Frost, D. Smith, S. May, N. Parkin, and D. Richman, abstr. 60, presented at the 14th Conference on Retroviruses and Opportunistic Infections, Los Angeles, CA, 25 to 28 February 2007). Among 900 recently diagnosed treatment-naive individuals with established infection from 10 U.S. cities, the prevalence of detectable resistance at 7.4% remains lower than even the most recent estimates among patients with primary infection (46
). Resistance prevalence estimates among untreated patients with chronic infection from Europe are quite similar, with 8.7% of patients with chronic infection (n
= 607) or unknown durations of infection (n
= 824) demonstrating resistance between 1996 and 2002 (47
). These data suggest that the prevalence of resistance declines with time after infection, resulting from the gradual replacement of DR virus by drug-sensitive virus until ultimately resistance testing fails to detect the resistant variant. Patients with chronic infection and older historical infection dates may predate the transmission of significant drug resistance or, if infected more recently (i.e., <10 years), may maintain some level of detectable drug resistance following transmission.
Transmitted DR variants were not associated with low RC values (mean, 87%). The mean RC was 89% for recently infected participants (PWT141) lacking any well-characterized drug resistance mutations (Fig. ). There was no difference in the mean RCs of the patients with transmitted DR virus who remained treatment naive, those who received antiretroviral treatment, and those who were infected with WT virus. Previous studies have demonstrated that DR virus is transmitted only about 20% as readily as drug-sensitive virus (21
). Among the subset of DR viruses that are transmitted, however, RC values are equivalent to those measured among newly infected individuals infected with WT virus, suggesting that the more fit DR viruses are more likely to be transmitted. This is consistent with the observation that the initial levels of viremia are comparable following acute infection with either DR or WT virus (12
). The fitness cost of individual RT resistance mutations is well established, particularly for 215Y, 184V, and 65R (5
). The transmission of any of the resistant variants more frequently associated with a fitness impairment might be associated with a more rapid reversion to a more “fit,” WT genotype that might fail to be detected by conventional sequencing (17
). Further studies are needed to determine whether these traditionally less fit variants are uniformly replaced at a significantly higher rate than other variants associated with higher RC values. Following transmission, the relatively high RC values associated with the majority of the transmitted resistant variants favor their persistence in the context of viral fitness conferred by the complete genome. This is in contrast to the observation in chronic infection where antiretroviral selective pressure selects for drug resistance mutations, which often reduce viral RC (23
Chronic HIV infection is characterized by a complex mixture of genetic variants, with approximately 75% of chronically infected individuals harboring DR forms (32
). The interruption of failing treatment has been associated with rapid emergence of more “fit” WT variants, typically over 12 to 16 weeks (7
). Since subjects infected with DR virus do not harbor WT variants at the time of infection, loss of detectable resistance requires mutational replacement of the DR virus by the WT virus rather than reemergence of a preexisting WT virus. The detection of complete shift from DR virus to drug-sensitive virus by bulk sequencing was demonstrated in only 1 of 14 patients, who were followed for a median of 2.1 years after the EDI. Using drug susceptibility as a marker of the proportion of resistant virus within the viral population in this patient, and based on the persistence of the K103K/N mixture, we estimated that the WT virus had less than a 1.5% fitness advantage compared to the K103N NNRTI-resistant variant. Given the relatively trivial fitness advantage of the transmitted WT virus compared to the transmitted DR virus, it is expected that most DR variants will persist, though the rate of replacement of mutations associated with larger changes in viral fitness (i.e., M184V) may be higher (43
). The persistence of 103K/N mixtures for over 2.6 years despite the short mutational distance between these mutants suggests that the mutation confers a small fitness cost consistent with high RC values. Given the low rate of replacement, it is impossible to estimate to what extent replacement times may vary between individuals due to factors such as host genetic background; however, if the few individuals in whom replacement of resistance occurred have higher rates of replacement, then the persistence of drug resistance may be even longer.
The persistence of drug resistance, however, does not imply the absence of ongoing genetic evolution. Substitutions occurred at sites not involved in drug resistance, presumably because of HIV-specific immune responses (10
). The rate of evolution at the amino acid level appeared to be independent of steady-state viremia (data not shown). The spontaneous appearance of drug resistance mutations in 3 of 14 patients (01-0512, 01-0550, and 01-0575) in the absence of selective drug pressure was most likely related to the presence of these particular resistant variants as relatively minor populations at the time of transmission, below the threshold of assay detection. More sensitive real-time PCR methods for detecting low-frequency minor variants among treatment-naive individuals have shown that resistant variants identified by real-time PCR and missed by conventional sequencing may represent 0.7% to 11% of the population by clonal sequencing (17
). Potential selective advantages may have resulted in the emergence of these resistant isolates over time to detectable levels despite the absence of selective drug pressure.
The persistence of DR variants provides a prolonged “window of opportunity” for secondary transmission of DR variants at a time when plasma viral load measures are routinely very high. Two subjects (study subject 01-0559 and a nonstudy subject) both acquired 103N as a secondary transmission from an untreated source subject (01-0512) 4 to 5 months after his EDI at a time when his viral load ranged from 5.5 to 6.3 log10
copies/ml. The same source subject, 3.2 years after his EDI and with a plasma viral load that remains stable at 5.0 log10
copies/ml, recently transmitted the same DR variant to yet another index subject. Of additional concern, the lower rates of viral turnover in the male genital tract result in even slower decay of HIV drug resistance in semen than in plasma (40
). The relative stability of drug resistance mutations within both source and recipient partners suggests that it is unlikely that we missed many replacement of resistance mutations by WT in the brief time between infection and study entry.
The persistence of transmitted drug resistance for years, prevalence estimates of transmitted drug resistance in newly infected individuals, and estimates of drug resistance among treatment-naive individuals with established infection all support the implementation of routine screening for primary drug resistance in all newly HIV-diagnosed, treatment-naive individuals. Primary resistance testing is estimated to be cost-effective in areas where the prevalence of resistance is greater than 4 to 5% (35
). The greater sensitivity of pol
genotype testing compared to phenotype testing in detecting drug resistance, coupled with its reduced cost, suggests that sequence-based resistance testing should be the resistance test of choice for newly diagnosed patients (38
). The health care implications of missing transmitted drug resistance are potentially significant. There are the costs of early treatment failures, the potential selection of additional DR variants in the setting of suboptimal treatment due to unrecognized resistance, and secondary transmissions that may continue for years. The relatively rapid emergence of drug resistance in the setting of treatment in a previously treatment-naive patient (infected for more than 5 to 7 years) should prompt the question as to whether initial infection with a DR variant may have been missed (Fig. ). Despite the apparent fitness “cost” of acquired drug resistance mutations in many treated patients, sexual transmission of HIV appears to select for the most fit DR variants when resistance is transmitted such that persistence of a highly replication-competent, DR variant is ensured for years in the new host. If proper records are maintained, a single genotype done for patients initiating care may alter the course of antiretroviral therapy initiated years later.
FIG. 7. Schematic illustration of the first decade (approximately) of HIV infection following infection with a resistant strain of virus. Patients initially infected with a DR variant will typically demonstrate a transient high-titer viremia, followed by a spontaneous (more ...)