Development of molecular methods for detection and epidemiological investigation of HIV-1, HIV-2, and HTLV-I/II infections

The work presented here was initiated to determine the possibilities of molecular methods for the detection and epidemiological investigation of HIV and HTLV infections. We present the results of a literature research and describe the development and partial evaluation of a new PCR method for the amplification of RNA and DNA sequences of the HIV-1 pol, env and gag, HIV-2 ltr and HTLV-I/II tax/rex genes. For the amplification of viral RNA, samples were treated with guanidium thiocyanate and sodium lauryl sarcosinate to disrupt the virus and to inactivate RNAses. Paramagnetic beads were used to extract the RNA, followed by solid-phase reverse transcription for cDNA synthesis. DNA or cDNA was amplified using a two-step PCR protocol in which the product from the first PCR was further amplified in a second PCR with nested primers combined with a touch-down temperature protocol to enhance sensitivity and specificity. A pilot study showed that in all peripheral blood mononuclear cell (PBMC) samples from seven HIV-1-infected individuals of CDC class II, proviral HIV-1 DNA was detected using three primer sets. HIV-1 RNA could be detected in the plasma from ten of fifteen HIV-1-infected individuals of CDC class II. Together with data from the literature, our results indicate that PCR methods are useful in the detection of HIV-1 infections and complementary to conventional methods such as enzyme immunoassays and Westernblot. They are especially useful when conventional methods do not allow a diagnosis to be made, for example in newborns of HIV-infected mothers, in monitoring of the viral load, and in patients with idiopathic CD4+ T-lymphocytopenia. Using the same method, we amplified HIV-2 and HTLV-I RNA and DNA sequences. However, clinical evaluation of these PCR methods must be conducted. This newly developed method may possibly be used in molecular epidemiological studies as we were able to directly sequence the product of the HIV-1 env np-PCR, the V3 variable region of the gp120 gene. However, molecular epidemiology must be conducted at the clonal level with more than one isolated part of the viral genome. For molecular epidemiological studies of HIV-1, for example, the variable regions V3, V4, and V5 of the gp120 gene together are useful targets. Promising methods for obtaining materials for PCR as well as serology with regard to epidemiology on the streets are the use of finger-prick blood spots on filter paper and saliva collection. Further studies are needed to determine the value of these methods in molecular epidemiological investigation