Evaluation of a Cost Effective In-House Method for HIV-1 Drug Resistance Genotyping Using Plasma Samples

<div><p>Objectives</p><p>Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.</p><p>Design</p><p>The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.</p><p>Methods</p><p>The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000–1.8 million RNA copies/ml.</p><p>Results</p><p>Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$vs 300$), thus making it cost effective.</p><p>Conclusions</p><p>The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.</p></div

Pairwise sequence identity analysis between the in-house and the ViroSeq method.

<p>Pairwise sequence identity analysis between the in-house and the ViroSeq method.</p

Primers used in the In-house method for HIV-1 drug resistance genotyping.

<p>Primers used in the In-house method for HIV-1 drug resistance genotyping.</p

Reproducibility and Precision of an in-house method.

<p>Reproducibility and Precision of an in-house method.</p

Phylogenetic analysis of the sequences generated during the evaluation of an in-house method.

<p>The phylogenetic tree was generated by using the PhyML software to create a maximum likelihood tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087441#pone.0087441-Chaturbhuj1" target="_blank">[13]</a>. The reference sequences were obtained from the Los Alamos HIV Database (<a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>). IHDR- sequences generated by an in-house method; VSQ- sequences generated by the ViroSeq method; R1 to R5- sample used in the reproducibility study (hilighted in blue); P1 to P5- sample used in the precision study (hilighted in red); A, B, C, D, E are the replicates of the same sample.</p

Drug resistance-associated amino acid positions in protease and reverse transcriptase from 26 proficiency testing panel plasma samples genotyped by the in-house and the ViroSeq methods.

<p>Note: Partial discordant positions are shown in bold.</p

Optimization of MS media for Callus and Suspension Culture of Costus pictus

Abstract-- Costus pictus is vulnerable species in India and threatened to extinction due to its indiscriminate collection. Tissue culture seems to be the way to preserve, protect and multiply this rare species for large scale multiplication. In the present study, protocol for callus culture was standardized using various concentrations of hormones (2, 4-D, Kinetin, IAA, BAP), MS medium (full strength, half strength, one-fourth strength), pH (4.5 – 7) and carbon sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol at 3 % and 6 % concentration). The highest callus growth was observed on half strength MS medium supplemented with 2,4-D / Kin (1/0.5 mg/L) and IAA/BAP (1 mg/L).Maximum callus growth was observed with glucose (3 % and 6%) and the pH value5.5. This is the first report of successful callus culture of Costus pictus leaves and also its suspension culture. The growth pattern of cell suspension culture was examined over a period of 10 days