Utility of HTLV proviral load quantification in diagnosis of HTLV-1-associated myelopathy requires international standardization

Grassi, Maria Fernanda Rios; Olavarria, Viviana Nilla; Kruschewsky, Ramon de Almeida; Yamano, Yoshihisa; Jacobson, Steven; Taylor, Graham P.; Martin, Fabiola; Galvão–Castro, Bernardo

doi:10.1016/j.jcv.2013.09.003

Cited by 16 publications

(11 citation statements)

References 15 publications

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“…IDH patients presented higher PVL than asymptomatic carriers of the same age range, as observed when compared with adult asymptomatic carriers [4]. Indeed, juvenile asymptomatic carriers presented PVL levels similar to adult asymptomatic carriers [11].…”

Section: Discussionsupporting

confidence: 56%

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HTLV-1 proviral load in infective dermatitis associated with HTLV-1 does not increase after the development of HTLV-1-associated myelopathy/tropical spastic paraparesis and does not decrease after IDH remission

Batista

Oliveira

Primo³

et al. 2019

PLoS Negl Trop Dis

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Section: Discussionsupporting

confidence: 56%

“…Considering that HAM/TSP in adults is characterized by high PVL [11], we expected that the association of HAM/TSP with IDH would be accompanied by an increase in PVL. However, we observed similar PVL in IDH patients with or without myelopathy.…”

Section: Discussionmentioning

confidence: 99%

HTLV-1 proviral load in infective dermatitis associated with HTLV-1 does not increase after the development of HTLV-1-associated myelopathy/tropical spastic paraparesis and does not decrease after IDH remission

Batista

Oliveira

Primo³

et al. 2019

PLoS Negl Trop Dis

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“…The coefficient of variation was low, less than 10 %, over a wide range of PVL. This favorably compares, if not exceeds, reliability of qPCR (Demontis et al 2013 ; Grassi et al 2013 ). Importantly, the coefficient of variation of ddPCR was lower than that of qPCR.…”

Section: Discussionmentioning

confidence: 66%

“…While the present study was not designed to demonstrate superiority of any PCR methodology, direct comparison of normalized HTLV-1 PVL measurements by ddPCR and qPCR showed a strong correlation between the two methods, although ddPCR demonstrated higher HTLV-1 PVL than qPCR. This may reflect differences in PCR assay platforms, different regions of HTLV-1 and housekeeping target gene amplification, and primer/probe design requirements (Grassi et al 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations

et al. 2014

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An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL—real-time PCR—has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe.

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“…Due to the variation of standardized techniques in different laboratories, there are limitations regarding the quantification of HTLV‐1 PVL (Grassi et al 2013). We hope to proceed towards the standardization of the HTLV‐1 PVL quantification technique.…”

Section: Discussionmentioning

confidence: 99%

Use of synthetic oligonucleotides for determination of HTLV‐1 proviral load by real‐time PCR: a helpful alternative approach in the clinical management

et al. 2020

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Aims: To evaluate the potential use of synthetic oligonucleotides as a standard curve for proviral load (PVL) of human T-cell leukaemia virus type 1 (HTLV-1) quantification in peripheral blood mononuclear cells (PBMC) of HTLV-1infected individuals by quantitative real-time polymerase chain reaction (qPCR) analysis. Methods and results: Synthetic oligonucleotides based on HTLV-1 genome were customized to use as a standard curve. Twelve anti-HTLV-1-positive samples with known HTLV-1 PVL, previously quantified by qPCR assay using TARL-2 cells as a conventional standard curve, were submitted to the new protocol. The proviral quantification levels had a high concordance with qPCR results using a conventional standard curve. The results demonstrate that the conventional standard curve can be replaced by a synthetic standard curve due to its ability to quantification based on the linearity and qPCR efficiency and similar results with a validated qPCR assay using a conventional standard curve. Conclusions: Synthetic oligonucleotides standard curves could be a very useful tool on HTLV-1 diagnosis and absolute HTLV-1 PVL quantification. Significance and Impact of the Study: HTLV-1 PVL determination using synthetic oligonucleotides standard curve by qPCR could be a helpful alternative for the laboratories that monitor infected patients as an important prognostic factor in HTLV-1-associated diseases progression. Also, it can decrease costs and overcome the biological limitations of the plasmid curve.

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Utility of HTLV proviral load quantification in diagnosis of HTLV-1-associated myelopathy requires international standardization

Cited by 16 publications

References 15 publications

HTLV-1 proviral load in infective dermatitis associated with HTLV-1 does not increase after the development of HTLV-1-associated myelopathy/tropical spastic paraparesis and does not decrease after IDH remission

HTLV-1 proviral load in infective dermatitis associated with HTLV-1 does not increase after the development of HTLV-1-associated myelopathy/tropical spastic paraparesis and does not decrease after IDH remission

Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations

Use of synthetic oligonucleotides for determination of HTLV‐1 proviral load by real‐time PCR: a helpful alternative approach in the clinical management

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