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20% Drug Resistance found in HIV Seroconverters Using Sensitive Assay
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"Detection of minor populations of drug-resistant HIV-1 in acute seroconverters"
"...Transmission of drug-resistant HIV-1 is a major health concern.... drug-resistant HIV-1 variants were detected in 10 of 49 acute seroconverter patients (20.4%) using a methodology to discriminate and quantify wild-type and drug-resistant variants, and to detect minor viral populations to levels below 1%.... Of the three mutations tested, M184V and K103N were found in six of 49 (12.2%) and five of 49 (10.2%) patients, respectively. In contrast, the L90M mutation was found in only one of 49 patients (2%)... Thus far, the impact of minor populations of drug-resistant HIV-1 on the response to antiretroviral therapy remains to be determined. However, the findings of several clinical studies suggest that minorities of drug-resistant HIV-1 could influence the clinical outcome of antiretroviral therapy. Minor drug-resistant variants have been detected especially in the early phase of therapy failure whereas these mutations were not seen by direct sequencing...."
AIDS: Volume 19(16) 4 November 2005
Metzner, Karin Ja; Rauch, Piaa; Walter, Haukea; Boesecke, Christophb; Zllner, Bernhardc; Jessen, Heikob; Schewe, Knudd; Fenske, Stefane; Gellermann, Holgere; Stellbrink, Hans-J rgenc
From the aUniversity of Erlangen-Nuremberg, Institute of Clinical and Molecular Virology, Erlangen
bPraxisgemeinschaft Jessen, Berlin
cUniversity Hospital Hamburg, Department of Medicine and Institute for Infection Medicine, Hamburg
dPraxis St. Georg, Hamburg
ePrivate Practices, Hamburg, Germany.
Abstract
Objective: The transmission of drug-resistant HIV-1 is a major health concern. To date, most clinical studies have relied on sequencing techniques for genotypic analyses which do not allow quantification of minority viral populations below 25%. As minor populations of drug-resistant HIV-1 could impact the efficiency of antiretroviral therapy, this study was performed to determine the prevalence of minor populations of drug-resistant HIV-1 in acute seroconverters.
Design and methods: Forty-nine acute seroconverters from two clinical centers in Germany were included in the study. Individuals were identified between June 1999 and March 2003, and none had received antiretroviral therapy prior to sampling. Minor populations of drug-resistant variants were detected by quantitative real-time polymerase chain reaction using allele-discriminating oligonucleotides for three key resistance mutations: L90M (protease), K103N and M184V (reverse transcriptase). The approximate discriminative power was between 0.01 and 0.2%.
Results:
- Drug-resistant variants were detected in 10 of 49 patients (20.4%).
- The L90M mutation was found in one of 49 (2%), the K103N mutation in five of 49 (10.2%) and the M184V mutation in six of 49 (12.2%) patients, respectively.
- In five of the 10 individuals with detectable drug-resistant virus (50%), the detected population represented a minor viral quasi-species (< 25% of viruses) and was not detected by direct sequencing.
Conclusions:
The prevalence of minor populations of drug-resistant HIV-1 in acute seroconverters can be frequently detected and may impact the success of antiretroviral therapy.
Introduction
Antiretroviral therapy (ART) has significantly reduced morbidity and mortality in HIV-infected individuals [1,2]. However, in a substantial fraction of treated patients, control of virus replication is incomplete or temporary, and resistance to antiretroviral drugs develops. The presence of drug-resistant HIV-1 is associated with impaired response to subsequent ART. After the first report of primary infection with drug-resistant HIV-1 [3], transmission of drug-resistant viral variants has been frequently observed (reviewed in [4]). In addition to the development of resistance during ART, transmission of drug-resistant HIV-1 has become a major health concern. Although a large number of studies aimed at quantifying HIV-1 drug-resistant variants have been performed, these studies are usually small in size and vary considerably in methodology and interpretation. Large, long-term studies are currently ongoing to determine whether the prevalence of drug-resistant viral variants in acute seroconverters is actually increasing (reviewed in [4]).
In general, the presence of HIV-1 drug resistance is evaluated by sequencing techniques that demonstrate the presence of typical mutations in the viral genome known to be associated with reduced sensitivity to an individual antiretroviral compound. These genotypic analyses are unable to detect and quantify minorities of drug-resistant viral quasi-species below 25% [5,6]. The clinical importance of minor populations of drug-resistant variants is still not clear. Certain clinical observations suggest that minor viral variants can emerge as the major viral population [7,8]. By using a methodology that allows the quantification of minor viral populations, we evaluated the prevalence of drug-resistant HIV-1, with emphasis on identification of drug-resistant variants as minor viral species in acutely HIV-infected patients.
Study design and patients
Patients referred to two clinical centers in Germany (Hamburg and Berlin) were included in this study. Acute seroconversion was confirmed as follows: At the first visit, all patients showed clinical signs and symptoms of an acute retroviral syndrome associated with positive plasma HIV-1 RNA levels. Most patients showed an incomplete pattern on western blot which changed to a complete pattern on subsequent visits. Patients with a complete pattern on western blot had a negative HIV-1 enzyme-linked immunosorbent assay test result within 3 months prior to presentation. Patients were identified between June 1999 and March 2003 and none had received ART before sampling. Study patients from Hamburg were participants in an observational cohort (HIV-AKUT study) that had been approved by the ethics committee of the Arztekammer Hamburg. Written informed consent was obtained from each patient prior to inclusion.
RESULTS
All 49 patients were tested for the presence of L90M, K103N and M184V mutations by quantitative real-time PCR for differential amplification. One or two drug-resistant mutations were found in nine of 49 (18.4%) patients. In one additional patient, as described below, one mutation was only detected by direct sequencing. The M184V mutation was detected in six of 49 (12.2%) at frequencies ranging from 1.6 to 38.1% in five patients who carried this mutation as a minor viral species. In only one patient the majority of viruses contained the M184V mutation (98.5 ± 0.1%). Genotypic analysis by conventional sequencing methods confirmed this result and also identified additional mutations associated with drug resistance to zidovudine in this patient (M41L and T215F, patient no. 3, data not shown). In four patients, viral populations carrying the K103N mutation were detected with prevalence from 9.78 to 99.5%. This mutation was present as a minor viral population in two patients (9.78 and 12.85%, respectively). In the remaining two patients, the K103N mutation was detected as the major viral quasi-species. The L90M mutation was found as a minor viral quasi-species in one patient (0.48 ± 0.05%, patient no. 35).
To confirm our results, sequencing was performed on viruses from all nine patients with evidence of drug-resistant mutations, and in five additional patients in which the L90M, K103N and M184V mutations were not detected. With the exception of patient no. 42, the presence of the mutations K103N and M184V mutations was confirmed by sequencing when these mutations were carried by the majority of viral species (patients no. 3, 35 and 47). Conversely, genotypic analysis by sequencing was unable to detect mutations present in a minority of viruses with the exception of patient no. 26 (Table 1). In this patient, sequencing revealed the presence of the K103N mutation, which represents a minor viral population (9.78 ± 0.1%) using quantitative real-time PCR for differential amplification. Sequence analysis in this patient revealed a transversion in codon 103 from AAA to AAT (amino acid change from K to N). The allele-discriminating oligonucleotides used in the quantitative real-time PCR assay were designed to distinguish between the wild-type codon AAA and the mutant codon AAC and were therefore unable to detect the AAA to AAT mutation. However, this means that a minor population in this patient is carrying the K103N mutation conferred by the codon AAC. In five patients for whom differential amplification by quantitative real-time PCR did not reveal any evidence of L90M, K103N or M184V mutations, these mutations were also not detected by direct sequencing, with the exception of patient no. 42. Furthermore in this patient, sequence analysis revealed a transversion in codon 103 from AAA to AAT (amino acid change from K to N) which was undetectable by the quantitative real-time PCR assay used for differential amplification.
Taken together, in 10 of 49 patients (20.4%) one or two tested mutations (L90M, K103N and M184V) were clearly detected as either minor or major viral populations. In all other patients, no evidence was found for these mutations within the range of detectable minor HIV-1 variants.
Discussion
Transmission of drug-resistant HIV-1 is a major health concern and currently large clinical studies are ongoing to determine the prevalence of drug-resistant viral variants in acute seroconverters (reviewed in [4]). In these studies, drug-resistant HIV-1 is detected by direct sequencing. A prevalence of 25% is regarded as the limit of detection of genotypic analysis by sequencing [5,6]. This limitation could lead to an underestimation of transmission of drug-resistant viral variants. Here, we investigated the prevalence of minor populations of drug-resistant HIV-1 variants in 49 acutely infected patients identified between June 1999 and March 2003. In this study cohort, minor populations of drug-resistant HIV-1 variants were detected by quantitative real-time PCR for differential amplification of three key resistance mutations: L90M (protease), K103N and M184V (reverse transcriptase).
Overall, drug-resistant HIV-1 variants were detected in 10 of 49 patients (20.4%). We did not observe a significant increase in the prevalence of drug-resistant HIV-1 over the 4-year period of observation for this study. Of the three mutations tested, M184V and K103N were found in six of 49 (12.2%) and five of 49 (10.2%) patients, respectively. In contrast, the L90M mutation was found in only one of 49 patients (2%). The M184V mutation confers high-level resistance to the commonly used antiretroviral drug lamivudine. Thus, it is not unexpected that the rapidly emerging single point mutation M184V is more frequently detected compared with the L90M mutation. Our finding of a high prevalence of the K103N mutation was surprising. It is known that resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) can be conferred by the K103N mutation alone, however, NNRTI-resistance is often accompanied by additional mutations, and K103N is not necessarily the first mutation to arise [11]. The high number of patients carrying the K103N mutation may therefore be a random effect due to the limited size of the present study.
In five of six patients carrying the M184V mutation this population represented a minority of viruses whereas in only one of five patients harboring the K103N mutation these viruses represented the minority. This may be due to differences in replicative capacity among drug-resistant viruses. It has been shown that the M184V mutation leads to a reduction of replicative capacity [12] whereas viral strains carrying the K103N mutation do not show an impaired replicative capacity compared with wild-type viruses [13]. Thus, wild-type variants quickly expand to represent the majority of the viral population when the drug-resistant variants are less replication competent. Of note, in those patients with a major population of drug-resistant virus the corresponding wild-type variant was also always detected as a minor population. Assuming wild-type viruses have an advantage in untreated patients, a change in the viral population must be expected. It has often been reported that drug-resistant HIV-1 persists after primary infection [14-17]. Thus far, only one study has shown a reversion to wild-type virus after transmission of a multi drug-resistant virus [18]. Therefore, it is conceivable that certain drug-resistant variants may have a replicative advantage over wild-type viruses.
We cannot exclude the possibility that minor viral populations of drug-resistant viruses are also present due to natural occurring diversity; namely, that these variants are spontaneously generated by random mutagenesis after primary infection. However, taking into account the reduced viral fitness described for viruses harbouring the M184V mutation [12], this seems unlikely, especially in patients with a prevalence of > 13% of viruses carrying the M184V mutation. It is more likely that patients infected by transmission of predominantly drug-resistant HIV-1 may experience a decrease in the prevalence of resistant viral variants in the absence of any selection pressure from antiretroviral drugs. This may be due to a presumed compromise in replicative capacity among drug-resistant viruses, such that wild-type variants quickly expand to represent the majority of the viral population.
Drug-resistant variants representing at least 25% of the viral population were found in four of the nine patients with detectable drug-resistant mutations (except patient no. 42). These patients would be expected to screen positive for drug-resistant variants using conventional sequencing [5,6] which was indeed the case for three of the four patients. In the fourth patient, approximately 38% of the viral population carried the M184V mutation, which was not detected by direct sequencing (Table 1, patient no. 41). Assuming transmission of wild-type viruses occurred in all patients in which drug-resistant minorities were not detected using quantitative real-time PCR for differential amplification (and with no additional sequencing information), this equals a transmission rate of drug-resistant viruses of 12.2% (six of 49 patients; the four patients mentioned above and the two additional patients carrying the K103N mutation as majority as revealed by sequencing). This is in close approximation of the currently estimated rate of transmission of drug-resistant viruses in studies using conventional sequencing for genotyping [4,19-21]. Of the nine cases in which resistant variants were detected by quantitative real-time PCR for differential amplification, only four were detected using conventional sequencing. No minor variants were detected by direct sequencing at a frequency of less than 38%. Thus, quantitative real-time PCR for differential amplification was slightly superior in screening for transmitted resistance and revealed a higher prevalence of drug-resistant viruses (20.4%), although only three key resistance mutations have been tested. Therefore, based on our findings, the transmission of drug-resistant HIV-1 variants is likely to be underestimated when determined solely by conventional sequencing methods.
Thus far, the impact of minor populations of drug-resistant HIV-1 on the response to antiretroviral therapy remains to be determined. However, the findings of several clinical studies suggest that minorities of drug-resistant HIV-1 could influence the clinical outcome of antiretroviral therapy. Minor drug-resistant variants have been detected especially in the early phase of therapy failure whereas these mutations were not seen by direct sequencing [7,22-24]. One study shows very nicely the kinetics of the emergence of drug-resistant viruses from minor to major viral populations in patients failing antiretroviral therapy [7]. It could be clinically beneficial to evaluate the prevalence of minor drug-resistant HIV-1 also in drug-naive patients such as acute seroconverters.
In summary, we report here the prevalence of drug-resistant viruses measured in acute seroconverters using a methodology to discriminate and quantify wild-type and drug-resistant variants, and to detect minor viral populations to levels below 1%. In a substantial proportion of patients with detectable drug-resistant viruses, this population represented a minority that was undetected by conventional sequencing techniques. Thus, the prevalence of drug-resistant HIV-1 as detected by sequencing seems likely to yield an underestimation of the transmission rate of these variant viruses. In the future, it may be advantageous to measure minor populations of drug-resistant viruses in acute seroconverters, even though the impact of these findings on the response to antiretroviral therapy remains to be determined.
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