ViveSTTM

ViveSTTM, formerly called SampleTankerTM (ST), is another dried sample storage and transportation device marketed by ViveBio Scientific (Alpharetta, GA, USA) since 2013 (https://vivebio.com/, accessed on 15 June 2022). The ST tubes have a proprietary, nonpaper-based absorbent matrix attached to the tube cap and a descant block at the bottom. The biological substance, such as proteins and nucleic acids, in the original liquid specimens can be retained on the matrix while the water part is evaporated during the drying process. The matrix can be rehydrated for downstream lab assays using molecular-grade water, and the reconstituted sample can then be recovered. Compared to 50~75 μL per DFPA spot and ~200 μL for the HemaSpot device, up to 1.5 mL of biological fluid can be loaded and dried onto the ST matrix and then stored or transported at ambient temperature. It is a revolutionary solution for collecting, storing, and transporting liquid specimens from the field. It aims to expand the decentralized collection of any liquid biological samples, including blood, plasma, serum, and other body fluid analytes.

Dried plasma specimens collected with the ST device have been validated for VL assays for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) [59–62], implying the application value of this new device in viral molecular assays. Lloyd et al. first reported ST application in HIVDRT during the XIII International HIV Drug Resistance Workshop in 2004 [60]. While lower VL readouts were obtained consistently from the ST plasma compared with frozen plasma, which was expected, the HIVDR mutations identified from the two compared analytes were concordant for all examined HIV-1 gene fragments. These observations were confirmed in an expanded study by this research group, which further demonstrated that the HIV viral RNA in ST plasma retained good integrity throughout the eight weeks of storage at 23 ◦C, 37 ◦C, and −80 ◦C [59].

In contrast, less optimistic findings were reported by Diallo et al., who assessed the application of the ST device for HIVDRT in RLS. They collected the whole blood or plasma specimens using the ST device and stored them at ambient temperature for 2 or 4 weeks. The performance of these two new analytes were compared against frozen plasma. Compared to 96% from frozen plasma specimens, both of the two new analytes performed poorly with significantly lower genotyping rates (48.98% and 42.85% for ST whole blood specimens stored for 2 and 4 weeks; 36% and 36% for ST plasma specimens stored for 2 and 4 weeks, respectively). Although the nucleotide sequence identities and the HIVDR profiles are highly concordant with the matching frozen plasmas for the successfully genotyped specimens, the low amplification rates from both ST specimens sugges<sup>t</sup> that the ST device may not be ideal for HIVDRT sample collection in RLS [63]. Similar conclusions were also drawn from a study by Kantor et al. in which the performance of two ST processed specimen types (STTM-plasma (STP) and STTM-blood (STB)) in HIV genotyping were assessed [64]. The HIVDRT success rates using STP and STB in the Kantor study were 32% and 12%, compared to 82% from matching frozen plasmas [64]. While the specimens in these studies

varied, the unfavorable outcomes from the two newer studies raise concerns over the preservation of HIV RNA/DNA integrity in the samples collected with ST tubes [63,64]. The further assessment of ViveST analytes for HIVDRT is warranted.

#### **3. Application Considerations on HIVDRT Analytes**

As described above, the quantity and quality of the HIV viral contents vary significantly among different analytes. The suitability of these analytes for the HIVDRT differ. The sensitivity, consistency, and accuracy vary considerably among the analytes due to the distinct nature of clinical analytes and the integrity of the HIV RNA or DNA templates they contain. Data collected from different analytes may hold differing values as applied to subsequent HIVDR interpretation. While plasma is the preferred analyte, it is not always available, especially in RLS, and alternative analytes are often required.

The suitability of an analyte for HIVDRT primarily depends on the resource availability for sample collection and transfer and downstream data application. One factor often neglected in HIVDRT analyte selection is the requirements from the downstream experimental procedures and data interpretation, which may differ significantly. Genotypic HIVDRT examines the presence of HIV DRMs either individually by allele-specific assays or collectively by sequencing using Sanger sequencing or NGS. Allele-specific assays target narrow viral genetic regions on which the impact from HIV RNA/DNA degradation is minimal. This is especially advantageous when a poorer HIV template quality is expected, such as the DFPAs and other dried analytes. In contrast, sequencing-based HIVDRT usually requires longer templates, making them more susceptible to HIV RNA/DNA degradation. One strategy to mitigate the limitation of degraded analytes, such as DFTAs, for HIVDRT is to implement modified protocols that generate shorter PCR amplicons or sequencing libraries. Compared to HIV RNA, a higher PCR amplification and sequencing success rate are expectable if DNA extracts from PBMC are used, especially when the VL is low. If targeting longer HIV genomic region(s) by long-range PCR or long-template NGS sequencing, fresh plasma/serum specimens with minimal HIV RNA/DNA degradation or HIV DNA extracts from PBMCs are always preferred.
