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Chapter 96 : Quantitation of Viremia and Determination of Drug Resistance in Patients with Human Immunodeficiency Virus Infection
Quantitation of Viremia and Determination of Drug Resistance in Patients with Human Immunodeficiency Virus Infection, Page 1 of 2< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815905/9781555813642_Chap96-1.gif /docserver/preview/fulltext/10.1128/9781555815905/9781555813642_Chap96-2.gif
In the current environment of human immunodeficiency virus type 1 (HIV-1) treatments under highly active antiretroviral therapy (HAART), not only an increase in the number of CD4+ T lymphocytes but also a decrease in the level of plasma viremia is expected; thus, it is essential to be able to quickly and accurately assess viral load. Continuous low-level viral replication, noncompliance issues, and gradual deterioration of the immune system have all contributed to the emergence of drug-resistant HIV-1. HIV drug resistance presents another challenge for clinicians. Until recently, the accepted approach to treating infected individuals with drug-resistant HIV-1 was thorough treatment history and viral load data. RNA is extracted by using guanidium thiocyanate and isopropanol. The use of an internal quantitation standard (IQS) monitors the efficiency of extraction and amplification and eliminates the need for a standard curve. The internal standard and the target sequence compete equally for primer binding and amplification in the PCR. Variables such as the efficiency of amplification and the number of cycles will have the same effect on both templates. The genotypic assays provide sequencing information that can be used to identify known resistance codons. Virus is concentrated from patient plasma by high-speed centrifugation, the virus pellet is lysed and viral RNA is purified using standard procedures.
Principle of the bDNA assay.
ICVPCR for the estimation of HIV-1 RNA in patient plasma. (A, B, and C) Different dilutions of mutant virus (VX-46) containing 0 pg (lane 1), 30 pg (lane 2), 3 pg (lane 3), 0.3 pg (lane 4), and 0.03 pg (lane 5) of p24 antigen were added to 100 μl aliquots of patients’ plasma. RNA isolation, cDNA synthesis, and PCR were carried out as described in the text. The sizes of DNA products from the mutant (148) and wild type (123) are shown on the right. Each panel is for plasma from one of three different patients. (D) Estimation of HIV-1 RNA in patients’ plasma. Patient 1 (triangles) had 10,000 copies, patient 2 (squares) had 7,600 copies, and patient 3 (circles) had 16,000 copies of RNA in 100 μl of plasma.
Structure of chimeric HIV. The PCR-amplified products are digested with restriction enzymes depending on the drug to be tested. To investigate drug resistance to protease inhibitor, RT inhibitor, or a combination of protease inhibitor and RT inhibitor, the products are digested with Apa1 plus Bal1, Bal1 plus PflIM1, or Apa1 plus PflM1. The digested products are ligated into pNLPFB backbone. LTR, long terminal repeat; aa, amino acid; PR, protease.