The reverse transcriptase (RT) enzyme is extracted from the virus particle in the Cavidi RT assay using a solid phase extraction manifold, and is quantified in a functional assay whereby RT synthesizes BrdU-DNA from a poly-A template bound to a 96-well plate [46
]. Synthesized DNA is then quantified using anti-BrdU conjugated to alkaline phosphatase followed by the addition of its substrate. The RT activity in the unknown sample is compared to that of a recombinant RT enzyme standard with a known concentration. The extrapolated result is reported as fg RT/ml of plasma or as HIV-1 RNA equivalents/ml using a conversion factor supplied by the manufacturer. The assay has recently undergone revisions to improve sensitivity [47
]. Since the assay measures a virion-associated enzyme, results are usually more comparable to plasma RNA [48
]. Additionally, since this is a functional assay for RT and does not rely on specific protein or nucleic acid sequences, it performs well when quantifying any HIV-1 subtype [49–51
]. A potential concern is that viruses with heavily mutated RT enzymes may be underestimated when compared to a recombinant wild-type RT enzyme, although preliminary studies suggest this may not be a significant problem [49
Five Key Papers on HIV Viral Load in Resource-Limited Settings
Majchrowicz, 2003 [10
] One of the first articles written describing the scope of the problem.
Patton et al., 2006 [45
] First description of the successful use of dried blood spots with the ultrasensitive p24 antigen assay.
Rouet et al., 2005 [62
] First demonstration of real-time PCR technology for monitoring and diagnosis in a resource-limited country.
Schupbach et al., 1996 [15
] An early paper comparing the ultrasensitive p24 antigen assay with HIV RNA viral load. Stevens et al., 2005 [25
] First independent comparison of the ultrasensitive p24 antigen assay and the Cavidi reverse transcriptase assay in a resource-limited country.
Since this assay is newer than the Ultra p24 antigen assay, fewer data are available and most studies have been conducted either by or in collaboration with the manufacturer. Stevens and colleagues [25
] compared the Roche RNA assay with both Ultra p24 antigen (external buffer) and the earlier version (version 1.0) of the Cavidi RT assay and found excellent correlation between RNA and RT results. The concentration of p24 antigen, RT, and RNA decreased in all patients after initiating ARV therapy, except in two individuals who had undetectable p24 and RT at baseline.
Crowe and colleagues have tested both the earlier version (version 1.0) and the more sensitive assay (version 2.0) in patients in Australia [49
]. The version 1.0 RT assay had detectable HIV-1 RT present in 98% of samples (n
= 127) with HIV-1 RNA >10,000 copies/ml. Sensitivity using the version 2.0 assay was higher, with detectable HIV RT in 95% of samples (n
= 69) with HIV RNA >1,000 copies/ml. A positive association was found between the log10
HIV RNA copies/ml and log10
HIV RT fg/ml variables using Pearson correlation (r
= 0.89, p
< 0.001; n
= 189 for version 1.0; r
= 0.89; n
= 85 for version 2.0). The RT activity closely followed the trend for HIV-1 RNA levels in samples (n
= 4–10 per patient) from 10 HIV-1 infected patients with progressive disease [48
]. Current (n
= 40), previous (n
= 30), or no (n
= 119) exposure to efavirenz had only a minor effect on the RT assay despite the tight binding of efavirenz to the HIV-1 RT enzyme. There was a 0.20 log10
decrease in viral load in the samples with efavirenz resistance mutations, as measured by RT when compared to RT polymerase chain reaction (PCR), indicating a possible decrease in RT fitness [49
Lombart and colleagues compared HIV-1 RNA (Roche COBAS Amplicor HIV-1 Monitor Test, version 1.5) with Ultra p24 antigen and the Cavidi RT assay, version 1.0 in samples from Burkina Faso [24
]. The RT assay detected 0%, 93%, and 100% of samples with viral loads of <10,000, 10,000–63,000, and >80,000 copies/ml, respectively. Seyoum and colleagues [50
] used the version 1.0 RT assay in a prospective study of 26 untreated patients with HIV in Ethiopia (subtype C), to compare results with the NucliSens QT assay. Although only 0.2 ml of plasma were used in this study instead of the 1.0 ml specified in the package insert, there was significant correlation between the two assays (n
= 178, r
= 0.65, p
< 0.001), and patient RT activity in general mirrored HIV-1 RNA changes. Sivapalasingam and colleagues [51
] compared results from the version 2.0 RT assay with the ultrasensitive Roche Amplicor Monitor HIV-1 RNA assay version 1.5 on plasma samples from patients in the United States (n
= 29, subtype B) and Cameroon (n
= 21, primarily CRF02_AG). They found that RT levels correlated significantly with plasma HIV-1 RNA viral loads using Spearman rank correlation (US: r
= 0.89; p
< 0.001; Cameroon: r
= 0.67, p
< 0.01). Among 32 samples with viral loads >2,000 copies/ml, 97% had detectable RT activity.