confer resistance to antiretroviral drugs are well characterized (Fig. 1) and there are a number of PCR-based assays for the specific detection of known point mutations.

Initially, allele-specific PCR assays were developed by incorporating the specific point mutation conferring resistance into the final codon of the PCR oligonucleotide primer, and using stringent annealing temperatures to inhibit PCR amplification unless a perfect match was obtained.[16] The disadvantages of this approach were that low levels of nonspecific amplification could not be ruled out and that each mutation required a specific primer pair. An alternative approach for characterizing mutations was to use point mutation assays (PMA) which captured PCR products containing either wild-type or mutant codons using oligonucleotides containing the codon of interest.[17] The Inno-LiPA HIV-1 resistance assay is based on reverse hybridization technology.[18] The Inno-LiPA strips allow the detection of limited mutations in RT and protease. The limitation of LiPA and other hybridization assays is that HIV-1 is extremely heterogeneous and as many as 40% of samples may fail to yield results which reduces the clinical benefit of the assay in the routine detection of resistance.

The gold standard approach to the detection of mutations that confer resistance to antiretroviral drugs is DNA sequencing. Sequencing has two limitations, firstly, it is time consuming and requires well-trained personnel, and, secondly, it is relatively insensitive, the mutant strain must constitute at least 25% of the sample. Not only are known mutations detected this way, but new mutations arising with novel combinations of therapy will also be detected. Of particular concern is the emergence of new mutations that may confer cross resistance to all compounds in a class, e.g., the Q151 M mutation affecting the reverse transcriptase gene confers cross resistance to all six nucleoside analogues in clinical use.[19] Current sequencing protocols require at least 1000 copies of HIV RNA per milliliter of plasma. However, nested PCR methods increase the sensitivity of the assay and sequences may be obtained from samples containing a plasma viral load of <50 copies/mL,[20] which enables earlier detection of treatment failure. Reproducibility between kits is good with Visible Genetics and Applied Biosystems (ABI) detecting the same mutations in 91% of samples tested although some reduction in the detection of mixtures of wild type/mutant was observed in the ABI system because of the limitations of the software.[21] Viroseq (ABI) was able to sequence 97% of samples from non-clade B HIV-1 strains from Uganda.[22]

Interpretation of sequencing should take into consideration the clinical history of the patient which may be complex in the case of transmission of resistant virus either from a partner who has been extensively treated with antiretroviral drugs or as the result of the casual transmission of resistant virus from an unnamed source. A useful tool in interpreting mutations in HIV sequences and for subtyping HIV clade is the University of Stanford database which may be accessed through http:// www.hivdb.stanford.edu. HIV subtype may also be determined via the National Centre for Biotechnology Information web site at http://www.ncbi.nlm.nih.gov/ retroviruses/subtype/subtype.html. Usefulness of individual interpretation systems depends upon the accuracy of the algorithms used to set up the system which differ from system to system and this affects the interpretation of the influence some mutations have on the sensitivity of individual drugs.

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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