HIV-1 RNA quantification from plasma spotted and dried onto fiiter papers compared to

iiquid plasma

3.1 Introduction

Acquired Immune Deficiency Syndrome (AIDS) is an immunosuppressive disorder characterised by depletion of the CD4 lymphocyte population. A progressive, severe immunodeficient state is heralded by a barrage of opportunistic infections and malignancies such as Kaposi's Sarcoma and Lymphomas. The causative agent, HIV-1 was first isolated in 1983 and HIV-2 in 1985. It is transmitted mainly through sex (homosexual and heterosexual), contaminated needles, mother to child transmission or contaminated blood and blood products.

The conventional method for detection of HIV infection is through serological identification of an immunologic response to the virus such as using enzyme linked immunosorbent assays and confirmation of results by western blots. However, ELISA and western blot have the drawback of depending on the immunological response which occasionally takes 6 months or more from the time of infection and have thus the inherent potential of missing the diagnosis in the perinatal period and primary HIV-1 infection.

The nucleic acid amplification techniques such as Reverse Transcriptase PCR, Roche Amplicor PCR and NAS BA (van Gemen et al. 1993) have an

advantage as they can detect the presence of the HlV-virus even before an antibody response occurs. Nucleic acid based assays detect the presence of HIV RNA and are generally more sensitive than p24 antigen assays (Van Gemen et al. 1993). It is now known that viral load measurements in the plasma ( Michael etal. 1995) and intracellular compartments (Schnittman et al. 1991; Piatak et al. 1993; Coombs 1994; Coombs et al. 1996 ) predict the rate of disease progression in HIV disease. A number of studies have now conclusively demonstrated that high levels of viral replication occur at all stages of the disease and that changes in viral RNA load are predictive of disease outcome (loannidis et al. 1996; Mellors et al. 1997) and response to therapy (Kappes et al. 1995; Katzenstein and Holodniy 1995). The above, coupled with the introduction of potent anti-retrovirals including the possibility of candidate vaccines, (Kitchen etal. 1995; Vella etal. 1995) have stimulated and re-emphasised the importance of viral load monitoring. The majority of the assays being used in the quantitaion of HIV-1 RNA require fresh samples of whole blood or liquid plasma or samples that have been cryo-preserved. The technology required to perform these tests is also complex, labour intensive and requires expensive equipment. The commonly used quantification assays measure HIV-1 virion RNA levels in plasma either by reverse transcriptase polymerase chain reaction (Katzenstein et al. 1994; Mulder ef a/. 1994; Cao etal. 1995) or the branched DNA signal amplification assay or culture (Cao et al 1995; PachI et al. 1995). It is therefore not surprising that the developing world with the bulk of the HIV burden, a staggering 22 million of global 33 million (UN AIDS 1999) cannot afford HIV-1 viral load monitoring assay due to cost and technical problems.

The ability to collect blood specimens on filter paper blots (Guthrie cards) provides a good, cheaper, reliable and convenient option for PCR based HIV- 1 studies (Cassol etal. 1991a; 1991b; Cassol etal. 1992a; 1992b; Cassol et al. 1996; Cassol et al. 1997). Using this technique, large numbers of difficult field specimens can be collected, dried and shipped without cryopreservation (Cassol et al. 1992b ). Filter paper application so far has been in genetic testing, HIV screening in new born (Cassol et al. 1994; Cassol et al. 1996), monitoring resistant patterns and sequencing and CD4 lymphocytes enumeration from dried blood spots (see chapter 2).

The HIV-1 epidemic consists of several 'mini' epidemics with different clades and genetic variants. To date, there have been relatively few large scale attempts to systematically characterise and track the spread of these rapidly emerging international variants. As a result, our knowledge of HIV-1 variation is based on a relatively small number of samples that have been haphazardly collected from a few easily accessed locations. An enhanced understanding of the frequency and distribution of HIV-1 global variants is crucial, not only to learn the origins and better understand the changing dynamics of the AIDS pandemic, but also to monitor the emergence of more virulent (or attenuated) strains, confirm that blood donor screening assays are sensitive and specific, and ensure vaccines are directed against biologically important, contemporary strains of HIV-1 that are prevalent within specific populations groups.

In Zambia as is the case in many developing countries, very little knowledge is available about the strains that are in the population but it is generally accepted that the clade C is prevalent (Betts et al 1997). The available assays for quantification of HIV-1 need to be re-evaluated in their performance against clade C strains as the primers used generally target the subtype B virus. The estimated cost of £40 per test of these assays have further compounded the situation and in Zambia today, no routine HIV-1 viral load quantitation can be available to the health institutions. Since current technology only allows quantification of HIV-1 RNA from liquid plasma, its use in the field studies will be restricted to the well financed research projects that can afford equipment allowing field site freezing, vapour phase tanks for transportation of samples and liquid nitrogen.

This section of the thesis was performed to evaluate whether the advantages conferred by the use of filter paper could be utilised and developed for use in the field.