Methodology

Antiviral drug resistance can be determined at the genotypic level by the detection of resistance mutations. Alternatively, it can be determined at the phenotypic level by measuring the ability of an HIV-1 isolate to grow in the presence of a drug, or by measuring the HIV-1 reverse transcriptase or protease enzyme activity in the presence of an inhibitor.

Sequencing is the most commonly used genotypic assay for the monitoring of resistance mutations. It provides information on all nucleotides of the sequenced region. The target sequence is amplified via polymerase chain reaction, and the sequence is determined based on the incorporation of dideoxynucleotides. At the moment, most clinical laboratories perform home-brew sequencing or they use one of the two commercially available sequencing assays [ViroSeq HIV-1 Genotyping System (Abbott) and Trugene HIV-1 Genotyping Kit (Bayer)]. Another option is to send samples to service laboratories (e.g., ViroLogic and Virco).

Presently, only three companies (ViroLogic, Virco, and VIRalliance) and a few national reference laboratories routinely perform replication-based phenotypic assays. These assays start with the amplification of the patient-derived HIV-1 target sequence via polymerase chain reaction. The amplification product is subsequently incorporated into a proviral laboratory clone via ligation or homologous recombination. This generates a stock of chimeric viruses. The viruses are tested for their ability to grow in the presence of different concentrations of inhibitors. The principle of chimeric viruses for the determination of the susceptibility profile is preferred over other phenotypic assays to obtain better reproducibility and automation possibilities.1-1,2-1

Resistance testing has to be performed on plasma samples before starting, stopping, or changing therapy. It is highly important to monitor the active replicating viral population responsible for the therapy failure, because it is the resistance pattern of this population that can be useful in understanding the observed therapy failure.

An important limitation of current available assays is their need for plasma samples with an HIV-1 viral load exceeding 1000 copies/mL because of the lower sensitivity of the cDNA synthesis and amplification procedures of long templates. This is required to achieve reliable results. Otherwise, nonrepresentative variants could be picked up because of stochastic events during the experimental procedures. However, switching therapy early after viral failure, when the HIV-1 viral load is detectable but still very low, results in better chance of response to the salvage therapy. There are only few data that provide the optimal point at which therapy should be changed in terms of long-term clinical outcome, but the short-term risk of any viral replication in the presence of inhibitors is the development of resistance.

Results of the resistance assays represent only the genotypic or phenotypic profile of the majority of quasi-species present in vivo because they have difficulties in detecting minor variants that reflect less than 10-50% of the total viral population.[3] It remains uncertain what the contribution of minor viral variants is to therapy failure.

As genotypic and phenotypic assays start with an amplification procedure, the sensitivity of both tests depends on the efficient hybridization of primers, and this is strongly influenced by the variability at the primer sites. Most of the assays have originally been optimized by using subtype B HIV-1 strains because such strains were responsible for the epidemic in developed countries.

However, in recent years, the prevalence of non-B subtypes has been increasing in Europe, and access to therapy has been extended to the developing world where non-B HIV-1 subtypes constitute the largest part of the epidemic.[4] Effort has been made to improve the performance of the assays on non-B HIV-1 subtypes by developing primers at more conserved sites or by the inclusion of backup primers, but no assay can guarantee to reproduce adequate results for all strains.

Both genotypic and phenotypic resistance assays have respective advantages and disadvantages (Table 1). Resistance assays have to be performed in highly specialized laboratory facilities. This particularly holds true for the phenotypic resistance assay because it requires biosafety level 2 or 3 facilities, whereas a genotypic resistance assay can be performed in a dedicated molecular biology laboratory. Because both types of assays require sophisticated technology and knowledge, a strict adherence to current laboratory standards is important; well-trained and experienced laboratory technicians are also essential.

Another practical consideration in the choice of resistance testing is the cost of the procedure. Phenotypic assays are still more expensive than genotypic assays.

Genotypic resistance assays can deliver results within a few days, whereas longer time is required to obtain

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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