Molecular methods for HIV-1 infection using dried blood-spot (DBS) for HIV-1 CRF01_AE subtypes have not been fully optimized. In this study assays for HIV-1 diagnosis or quantitation were evaluated using infant DBS from Thailand. Paired DBS and whole blood samples from 56 HIV-1 CRF01_AE or B'-infected infants were tested for infant diagnosis using modified Amplicor DNA PCR and NucliSens RNA NASBA and an in-house real-time PCR assay. The Amplicor Monitor viral load (VL) assay, with modifications for DBS, was also evaluated. DBS VL were hematocrit corrected. Stability studies were done on DBS stored at -70 degrees C to 37 degrees C for up to 1 year. The DBS diagnostic assays were 96-100% sensitive and 100% specific for HIV-1 diagnosis. DBS HIV-1 VL were highly correlated with plasma VL when corrected using the actual or an assumed hematocrit factor (r(c)=0.88 or 0.93, respectively). HIV-1 DNA in DBS appeared to be more stable than RNA and could be detected after up to 9 months at most temperatures. DBS VL could be consistently determined when stored frozen. These results show that DBS can be used accurately instead of whole blood for the diagnosis of HIV-1 infection and VL quantitation, particularly if samples are appropriately stored.
"The rate found was higher than the one reported by Charpentier and colleagues (46%) [Charpentier et al., 2011]. Differences in amplification success rate could probably due to shipping conditions and conservation of DBS and on also to the extraction of nucleic acids and HIV-1 genetic diversity [Leelawiwat et al., 2009; WHO, 2012]. In this study, virological failure observed in 55/231 (23.8%) cases in decentralized settings is two fold higher than that described in Dakar [Aghokeng et al., 2011]. "
"DBS and DPS have been shown to be useful means for specimen transportation and preservation for several RNA viruses (Chibo, et al. 2005; Choi, et al., 2009; De Swart, et al., 2001; de Crignis, et al, 2010; Desbois, et al., 2009; Katz, et al., 2002; Lenselink, et al., 2009). They have been shown to support quantitative HIV viral load testing for monitoring patients on antiretroviral therapy (ART) (Andreotti, et al., 2010; Garrido, et al., 2009; Johannessen, et al., 2009a; Leelawiwat, et al., 2009; Lofgren, et al., 2009; Marconi, et al., 2009; Mbida, et al., 2009; Mehta, et al., 2009; Reigadas, et al., 2009; Viljoen, et al., 2010). However, DBS and DPS place constraints on the lower limit of detection (LOD) attainable and the dynamic range over which there is adequate quantitative discrimination (Andreotti, et al., 2010; Johannessen, et al., 2009a; Johannessen, et al., 2009b; Lofgren, et al., 2009). "
[Show abstract][Hide abstract] ABSTRACT: Two approaches to stabilize viral nucleic acid in processed clinical specimens were evaluated. HIV-1 RNA extracted from clinical specimens was stabilized in a dry matrix in a commercial product (RNAstable, Biomatrica, San Diego, CA, USA) and in a reverse-transcription reaction mixture in liquid form as cDNA. As few as 145 HIV-1 genome copies of viral RNA are reliably stabilized by RNAstable at 45°C for 92 days and in the cDNA format at 45°C for 7 days as determined by real-time PCR. With RNAstable the R(2) at days 1, 7, and 92 were 0.888, 0.871, and 0.943 when compared to baseline viral load values. The cDNA generated from the same clinical specimens was highly stable with an R(2) value of 0.762 when comparing viral load determinations at day 7 to baseline values. In conclusion viral RNA stabilized in a dry RNAstable matrix is highly stable for long periods of time at high temperatures across a substantial dynamic range. Viral RNA signal can also be stabilized in liquid in the form of cDNA for limited periods of time. Methods that reduce reliance on the cold chain and preserve specimen integrity are critical for extending the reach of molecular testing to low-resource settings. Products based on anhydrobiosis, such as the RNAstable should be evaluated further to support viral pathogen diagnosis.
"Several commercial assays are capable of measuring HIV-1 VL across a range of diverse HIV-1 variants (Valasek and Repa, 2005) (Roche Amplicor), but these assays require plasma (typically requiring a " cold chain " of storage) and reagent costs of approximately US $50.00 per sample. These requirements have restricted their use in resource-limited settings (Creek et al., 2007; Leelawiwat et al., 2009; Ou et al., 2007; Sherman et al., 2005; Uttayamakul et al., 2005). Less costly PCR-based in-house assays have been described, but these do not all contend with the genetic diversity of HIV-1, especially given the complex and evolving subtype composition in many regions of the world. "
[Show abstract][Hide abstract] ABSTRACT: There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (10(1)-10(7) copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 10(5) cells), and broad applicability to a range of HIV-1 subtypes (including A, B, C, D, F, H, B/C, and A/E CRFs). Using the LNA™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r=0.765, p<0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate.
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