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PLoS One. 2014 Mar 6;9(3):e89826. doi: 10.1371/journal.pone.0089826. eCollection 2014.

Clinical evaluation of a low cost, in-house developed real-time RT-PCR human immunodeficiency virus type 1 (HIV-1) quantitation assay for HIV-1 infected patients.

Author information

  • 1Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
  • 2Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore, Singapore.
  • 3Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore; Department of Paediatrics, University Children's Medical Institute, National University Hospital, Singapore.
  • 4Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore, Singapore.
  • 5Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore; Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
  • 6Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore; Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Lee Kong Chian School Of Medicine, Nanyang Technological University, Singapore.

Abstract

OBJECTIVES:

HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.

MATERIALS AND METHODS:

A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays.

RESULTS:

The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100-1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A-H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate.

CONCLUSIONS:

With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.

PMID:
24603460
[PubMed - in process]
PMCID:
PMC3945479
Free PMC Article
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