Alternative to Dried Blood Spot Analysis

The U.S. President’s Emergency Plan for AIDS Relief, in conjunction with the Global Fund has initiated antiretroviral therapy (ART) for 3.7 million patients in the developing world as of 2009. However, the distribution of therapy has outpaced the ability of most countries to monitor patients for treatment failure. Two diagnostic tests are performed to assess therapy effectiveness: CD4 cell counts and viral load tests, also called nucleic acid tests (NATs).  Although still debated, several studies have concluded that for patients on ART, CD4 counts alone are not sufficient to justify therapy modifications, and when feasible, a combination of both tests or NATs alone is favored(1).

NATs are significantly more complicated to perform than CD4 counts. Both tests require a blood sample, but NATs require several additional sample preparation steps to extract HIV RNA. Once RNA is isolated and purified, it must be amplified by a nucleic acid amplification technique like PCR. NAT protocols are time consuming and almost always carried out at a central laboratory in low resource settings due to concerns about cross contamination and quality control.  Results are routed back to the patient via the primary care provider, who makes treatment modification decisions.

Many studies have focused on decreasing the cost of NATs(2). Two main strategies for improvement have emerged, 1) moving the entire assay to the point-of-care (POC) through the development and use of small, portable, and simple diagnostic platforms, and 2) developing sample collection technologies that reduce or eliminate the need for a cold chain for storage and transport.  While the first strategy represents a complete long-term solution, it may not be appropriate for all settings. The second strategy requires much less technology development and is closer to implementation. Here, I describe two competing non-traditional sample collection technologies.

Current Sample Collection Procedures

There are several commercial NAT kits available for HIV(3). Each includes a companion sample preparation protocol, and in some cases a distinct sample preparation instrument. Blood is usually collected at a satellite collection site (SCS) via venipuncture and shipped to the testing laboratory. At the test lab, plasma is separated from the blood using centrifugation, and RNA isolation is performed using bench top techniques and equipment. Once RNA is isolated from plasma, nucleic acid amplification is performed, followed by an optical readout.  The kits are highly sensitive and specific.  The kit and associated sample preparation steps are borne by the central lab and the cost per test varies, roughly $80-$130 per result delivered to the patient(2, 4). The cost of the test and associated sample preparation represents about 80% of the total cost per result.  Patient costs in travel, food, lodging and opportunity costs is ~10%, and costs to the SCS are ~10%.

Removing the cold chain would greatly reduce costs to the patient and the SCS.  If the collected samples do not require a cold chain, then sample collection can be carried out closer to the patient, thus reducing travel and opportunity costs. If the SCS does not have to maintain special storage on site, the samples can be collected, batched and sent on a regular schedule to the central testing facility, thus reducing costs to the SCS.  If the sample collection method also prepares the samplefor direct input to a NAT, then significant costs can be offset at the central lab facility.

Alternative Collection Methods

Dried blood spot (DBS) collection has been used widely for qualitative (yes/no) HIV diagnosis, and is routinely used for infant screening(5).  A finger prick blood sample is used instead of venipuncture. A drop of blood 50-100 microliters is dropped onto a piece of Whatman 903 filter paper. The paper is dried and packaged with a desiccant for shipping. In this condition, the nucleic acids are preserved to a varying degree, depending on storage conditions, and the virus is inactivated(6, 7). The paper is shipped to the central testing site where RNA is extracted for downstream testing. DBS collection saves money for the patient and the SCS, since very little equipment is needed, little training is needed, and the collection can be performed anywhere. The limitations of DBS methods are still under investigation, but a lower limit of detection of ~4000 copies/ml for an RNA viral load test is supported by several recent studies(6-8). Whether this limit is sufficient to detect ART failure depends on the local definition of treatment failure.

Minimally Instrumented Sample Collection

In our laboratory we have developed a no-power instrument for field use to collect a patient sample, isolate and stabilize RNA, and stabilize the sample for storage and shipping(9, 10).  Sample collection can be either finger prick (microliters) or venipuncture (milliliter) samples.  Samples are mixed with lysis buffer upon collection and introduced into a pressure driven extraction apparatus. The extracted sample is washed, treated with an RNA preservation reagent and dried, all using air pressure provided by an attached mechanical bicycle pump. No external power source is needed. The method differs from the DBS method in two important ways, 1) the RNA isextracted and stabilized at the POC, and 2) larger input samples are possible, thus driving down the limit of detection. While neither method requires a cold chain for transport to the central lab, the instrumented method delivers a prepared sample that only needs to be eluted in water before the NAT. This method requires more user training than DBS, and the health worker performs more individual steps. The instrumented method lowers cost for the patient and the central laboratory. The costs at the SCS will likely remain the same due to increased training needs.

Your input

We would love to hear from folks in the field about what kind of sample collection system would work best for you. What are your needs? What limits of detection do you require? How much training is appropriate? We look forward to the discussion!

References

1.         L. Lynen, J. Van Griensven, J. Elliott, Curr Opin HIV AIDS 5, 1 (Jan, 2010).

2.         A. Puren, J. L. Gerlach, B. H. Weigl, D. M. Kelso, G. J. Domingo,  J Infect Dis 201 Suppl 1, S27 (Apr 15, 2010).

3.         S. Butto, B. Suligoi, E. Fanales-Belasio, M. Raimondo,Ann Ist Super Sanita 46, 24 (2010).

4.         J. Gerlach et al.,  J Int AIDS Soc 13, 43 (2010).

5.         A. Johannessen, Bioanalysis 2, 1893 (Nov, 2010).

6.         W. Leelawiwat et al.,  J Virol Methods 155, 109 (Feb, 2009).

7.         A. Johannessen, M. Troseid, A. Calmy, J Antimicrob Chemother 64, 1126 (Dec, 2009).

8.         R. L. Hamers, P. W. Smit, W. Stevens, R. Schuurman, T. F. Rinke de Wit,  Antivir Ther 14, 619 (2009).

9.         J. Chu, in MIT Technolgy Review. (Cambridge, MA, 2009).

10.       A. Bhattacharyya and C. M. Klapperich, Conf Proc IEEE Eng Med Biol Soc 2009, 1067 (2009).