Development of a Qualitative Quantitative Polymerase Chain Reaction Test to Identify Patients Failing First-Line Therapy to Non-Nucleotide Reverse Transcriptase Inhibitor.
Antiretroviral therapy (ART) can be compromised by selection of drug resistance strains, which can be promoted by lack of adherence during therapy and drug tolerance, and some of these drug-resistant strains can persist for years as minority populations. The K103N drug resistance mutation is selected by the use of non-nucleotide reverse transcriptase inhibitors, including nevirapine or efavirenz (EFV), used in low-income countries. Here we describe the use of a less expensive qualitative point mutation polymerase chain reaction (PMqPCRK103N) targeting K103N mutation. To validate the use of this methodology, we tested previously sequenced samples from patients treated with highly active ART with viral loads above 2,000 copies/ml and compared the results of our assay with Illumina deep sequencing. Due to its low cost and high specificity, this test is particularly suitable for low-income countries to screen for pretreatment resistance in patients either initiating ART or failing first-line regimens containing EFV.
1 Faculdade de Farmacia, Universidade Federal do Rio de Janeiro (UFRJ) , Rio de Janeiro, Brazil .
Sergio
S
L Machado
2 Laboratório de Virologia Molecular, Instituto de Biologia, Departamento de Genética, Universidade Federal do Rio de Janeiro (UFRJ) , Rio de Janeiro, Brazil .
Gabriel S
GS
Gonçalves
3 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison , Madison, Wisconsin.
Dawn
D
Dudley
3 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison , Madison, Wisconsin.
David
D
O'Connor
1 Faculdade de Farmacia, Universidade Federal do Rio de Janeiro (UFRJ) , Rio de Janeiro, Brazil .
Helena
H
Keiko Toma
4 Laboratorio de AIDS and Imunologia Molecular , Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brazil .
José Carlos Couto
JC
Fernandes
2 Laboratório de Virologia Molecular, Instituto de Biologia, Departamento de Genética, Universidade Federal do Rio de Janeiro (UFRJ) , Rio de Janeiro, Brazil .
Amilcar
A
Tanuri
D000480:Alkynes; D048588:Benzoxazines / Q000627:therapeutic use; D003365:Costs and Cost Analysis; D003521:Cyclopropanes; D016903:Drug Monitoring / Q000191:economics / Q000379:methods*; D024882:Drug Resistance, Viral; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods; D015658:HIV Infections / Q000175:diagnosis* / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D020125:Mutation, Missense; D019829:Nevirapine / Q000627:therapeutic use; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*; D012680:Sensitivity and Specificity; D017211:Treatment Failure
D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; C098320:efavirenz
Illumina deep sequencing; K103N; NNRTI; SYBR Green; Sanger; low-frequency DRM; low-income countries; qPCR