Dried blood spots can be used to measure HIV viral load, using the Abbott RealTime HIV-1 assay, although they tend to produce somewhat higher values than viral load tests in plasma, and the method may fail to quantify the virus in some specimens from subjects with low plasma viral loads, according to South African and international studies described at the Fourth South African AIDS Conference in Durban.
In a study with 497 samples, the method would detect 94% of viral loads below 10,000 copies/ml, according to Professor Lesley Scott of the Department of Molecular Medicine and Haematology at the University of the Witwatersrand.
While still not perfect, these results suggest that the method does have potential to be used to confirm antiretroviral treatment failure virologically in situations with limited access to plasma viral load measurement because of technical or logistical constraints.
Scaling up laboratory monitoring for people on antiretroviral therapy (ART) in resource-limited settings
In the South African national programme, about 560,000 people with HIV on ART currently require viral load testing at least twice a year. The test is no longer used for staging disease in people not yet on ART. Rather, it is used to monitor whether patients have failed on first-line treatment (defined as a viral load over 5000 copies/ml) and need to be switched to a second-line drug regimen.
Laboratory testing in the public sector is currently performed by the National Health Laboratory Service (NHLS) using nucleic acid testing. Out of 265 laboratories in South Africa, only 17 perform viral load testing.
There are a number of challenges in rolling out viral load testing in South Africa, including: the high sample volumes, that will only increase as the programme grows; the logistics of transporting specimens from remote sites; costs (of the equipment, maintenance, supplies, labour); phlebotomy, which is particularly difficult in children; a shortage of qualified technicians; and the limited sample throughput (number of tests that can be performed at once) using the different technology platforms.
The South African National Health Laboratory Services have responded by trying to increase throughput, develop more automated and central laboratories, and improve sample collection, transport and storage techniques. They have also evaluated alternative viral load testing technologies – an example of which is using flow cytometry as a screening technology to reduce testing volumes for viral load.
“And of course – the 'Holy Grail' is that we would all like to have point of care [POC] testing of viral loads in the clinics,” said Prof. Scott.
Dried blood spots
Viral load testing of dried blood spots (DBS) could help address some of the problems with sample collection, transport and storage. DBS are increasingly being used for the diagnosis of HIV in infants because they can easily be collected from infants, packaged and sent to a centralised lab for testing. But all that is required for diagnosis is to detect HIV in the specimen – which is much easier than reliably measuring how much virus there is.
“The big research question is: can DBS [be used to] measure at least 5000 copies/ml for monitoring?” said Prof. Scott.
To find out, Professor Scott looked at the Realtime HIV-1 assay, a commercial platform that has recently been introduced for plasma viral load testing in South Africa. [This section describes the methods for a laboratory audience.]
The platform has an extraction stage, which is automated, and a real-time detection (m2000rt) system that is based on fluorescence. In contrast to other viral load tests, an advantage of this system is that it can be performed in a single room. On plasma specimens, the assay has a very broad linear range: from 40 copies to 10 million copies/ml with a fairly high throughput of 93 samples per day per run, more with overnight testing.
To use DBS, 50 µl per spot from treated blood were collected on Whatman Protein Saver 903 cards within 24 hours post-venesection. These were manually punched into 50 ml Nunc tubes to prevent any further handling. The DBS was stored in the desiccant for two weeks at room temperature before being tested and the results compared to those with 1 ml of plasma. Two spots (100 µl) were used in the extraction.
Professor Scott and colleagues performed a pilot study to determine the correct extraction requirement and found an hour’s incubation in 1.7 ml lysis buffer was adequate.
Testing 21 samples (previously stored at -20ºC), and comparing them with viral load measurements of plasma samples obtained from the same patients, they found that, in the majority of the samples, DBS viral loads actually yielded a higher value, which was surprising because the amount of blood sampled was very low. This would normally be expected to result in a less sensitive reading. However, the mean difference was 0.2 log, with a lower standard deviation.
But most remote sites can’t refrigerate their specimens. So a bigger study was performed with 98 DBS samples stored at room temperature, again compared with plasma samples drawn from the same patients.
Viral load measurements could be obtained on 85 of these, with values ranging from 1.6 to 6.82 log copies/ml. In these specimens, DBS still gave viral load values higher than in plasma, though again the mean difference was within an acceptable range. However, some of the specimens were outliers: 20 (23%) had values that were over 0.5 log higher, and six (7.1%) had values that were more than 1 log higher than in plasma. But of the latter six, all had absolute values of less than 4 log copies/ml with DBS viral load.
“It’s interesting to note that this assay can actually generate results down to the lower level and actually yield a higher value,” said Professor Scott.
Viral load was undetectable in 13 of the samples, five of which were also undetectable on plasma viral load. The eight remaining had values ranging from 40 copies/ml to 546 copies/ml with plasma viral load. Thus, anything lower than 500 should be considered undetectable with this method.
Prof. Scott was also involved in an international study of 497 samples that were tested at eight other international sites. Again, there were outliers, again in the lower range, and DBS generally produced a higher viral load measure than plasma but with mean acceptable differences, according to Professor Scott.
“If you had to ask what are the sensitivities of the DBS to provide viral load results, in the range between 400 to 1,000 copies/ml, at least 70% of the DBS values could generate those results. This extended to at least 94% of viral load results given in plasma values that had less than 10,000 copies/ml,” she said.
A little more experience may have to be gained with this methodology to figure out what to make of the higher values among the outliers – particularly when those differences might determine whether a person is switched to second-line therapy or not. Under the South African guidelines, a half log higher value could make a difference if the person has a viral load close to 5000 copies/ml already – many other settings, however, use a 10,000 copies/ml cut-off.
Professor Scott is investigating whether the higher values might be due to the test picking up proviral HIV DNA which comes from the whole blood on the DBS.
But, with a little more refinement, DBS viral loads (using this platform at least) may offer a way to perform viral loads for patients at remote clinics that cannot access a labora-tory. Furthermore, it may be possible to outfit more of the laboratories in South Africa with these machines and increase monitoring capacity.
At present, the SA NHLS must perform at least 1.2 million viral load tests per annum for the current number of individuals on treatment, and the demands for viral load scale-up will keep growing. Later in the conference, Dr Terry Marshall of the NHLS gave an upbeat forecast of the programme’s ability to keep scaling up – the service has indeed grown at an astounding pace.
But in a press conference afterwards, Dr Marshall said that while they were hopeful about DBS viral loads, specimen collection is trickier than when DBS are collected for infant diagnosis.
“The problem with quantitating from a dried blood spot is that you need exactly the right amount of blood to be placed on the spot at the time of collection. With the DBS in infants, we have a lot of ‘fun’ already. We get all sorts of variability occurring [in the size of the sample] already, sometimes just a tiny smear on the paper, which you cannot analyse. So it’s something that is very very doable, but it does involve very very careful specimen collection and I anticipate that we will have our greatest challenge in getting the clinics consistently trained up to the point where we will always get good quality specimens,” she said.
Scott L. Dried blood spot (DBS) HIV viral load testing using the Abbott Realtime HIV-1 assay. Fourth South African AIDS Conference, Durban, abstract 239, 2009.