title: 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.
abstract: OBJECTIVE
  HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.
  
  
  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.
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Palvinder
authors.initials: P
authors.lastname: Kaur
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Wei Xin
authors.initials: WX
authors.lastname: Khong
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore, Singapore.
authors.forename: Sue Yuen
authors.initials: SY
authors.lastname: Wee
authors.affiliation: Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore; Department of Paediatrics, University Children's Medical Institute, National University Hospital, Singapore.
authors.forename: Eng Lee
authors.initials: EL
authors.lastname: Tan
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore, Singapore.
authors.forename: Juergen
authors.initials: J
authors.lastname: Pipper
authors.affiliation: Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore; Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Koay
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Kah Ying
authors.initials: KY
authors.lastname: Ng
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Joe Kwan
authors.initials: JK
authors.lastname: Yap
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Kuan Kiat
authors.initials: KK
authors.lastname: Chew
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Mei Ting
authors.initials: MT
authors.lastname: Tan
authors.affiliation: Institute 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.
authors.forename: Yee Sin
authors.initials: YS
authors.lastname: Leo
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore, Singapore.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Inoue
authors.affiliation: Institute of Infectious Disease and Epidemiology, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore.
authors.forename: Oon Tek
authors.initials: OT
authors.lastname: Ng
mesh_terms: D003362:Cost-Benefit Analysis; D015658:HIV Infections / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000187:drug effects; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D011933:Reagent Kits, Diagnostic / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D012846:Singapore; D019562:Viral Load; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D054301:gag Gene Products, Human Immunodeficiency Virus