Lofgren et al.  recently reported their experiences with the use of dried blood spots (DBS) for quantification of HIV-1 RNA in a clinical setting in Tanzania. Although results obtained with DBS were highly sensitive and specific to diagnose infant HIV infection, DBS results were less specific to detect treatment failure in patients who received antiretroviral treatment (ART). In the latter group, there were a number of false-positive results in DBS, particularly among patients with low-level viraemia (<3 log10 copies/ml) in plasma. The same problem has also been reported by others when DBS are used to monitor viral load [2,3].
DBS consist of whole blood, and therefore proviral DNA from peripheral blood mononuclear cells can contribute in the amplification process. Most commercial viral load assays are based on the reverse transcriptase (RT)-PCR, which amplifies all nucleic acid material in the sample. A positive amplification result obtained when DBS are used instead of cell-free plasma can therefore be caused by either RNA or DNA or both. However, the presence of HIV-1 DNA in a patient sample does not imply treatment failure, and joint amplification of RNA and DNA therefore leads to serious problems in the interpretation of a positive result.
This problem can be overcome by using a viral load assay based on nucleic acid sequence-based amplification (NASBA), such as the commercial NucliSENS EasyQ HIV-1 assay (BioMérieux Inc., Marcy l'Etoile, France). The NASBA method is an isothermal amplification (41°C), which specifically amplifies single-stranded RNA, by the use of T7 RNA polymerase. In the absence of heat denaturation, double-stranded DNA cannot participate in the amplification process [4,5]. NASBA technology, therefore, is particularly suitable when DBS are used instead of cell-free plasma for HIV-1 RNA quantification.
Recent experiences with the use of NASBA-based assays for viral load quantification on DBS have been promising. Garrido et al.  compared HIV-1 RNA levels in plasma and DBS using the NucliSENS EasyQ HIV-1 assay. Overall, there was a strong correlation between plasma and DBS viral loads (R 2 = 0.87), and in only two of 97 specimens did the DBS results exceed the plasma result by more than 0.5 log10 copies/ml . Furthermore, in our own study from rural Tanzania, we obtained similar good results using DBS specimens obtained under field conditions. The correlation between plasma and DBS viral loads was strong (R 2 = 0.75) and, among 33 specimens with undetectable viraemia in plasma, only two had detectable HIV-1 RNA in DBS using the NucliSENS EasyQ HIV-1 assay . Although the specificity was high in both these studies, the sensitivity was reduced in patients with low-level viraemia (<3.5 log10 copies/ml) because of the small volume of blood per dried spot.
There is an urgent need for a reliable strategy to identify those in need of second-line ART in resource-limited settings. Clinical and immunological criteria, as recommended by the World Health Organization, have poor sensitivity and specificity to detect virological failure . On the other hand, viral load measurements and genotypic resistance testing, as recommended in industrialized countries, are rarely available because of high costs and stringent requirements for the storage and shipment of plasma.
DBS can be stored and shipped at ambient temperature and may be a convenient alternative to plasma in remote resource-limited settings. Lofgren et al.  demonstrated the feasibility of using DBS for viral load monitoring at two rural healthcare facilities in Tanzania. The reduced sensitivity of DBS when viral burden is low might be acceptable, as a first-line antiretroviral regimen can be efficacious for years in spite of low-level viraemia . On the contrary, a high number of false-positive results, as reported by Lofgren et al. , would be of major concern, as it would lead to a premature switch to costly and complex second-line ART in a number of patients with adequate virological suppression. Thus, in order to avoid overdiagnosis of treatment failure, DBS should be used in combination with a NASBA-based assay, rather than an RT-PCR-based assay, for the reliable quantification of HIV-1 RNA.
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