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Journal of Clinical Microbiology, February 2007, p. 517-521, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.02016-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation of Dried Blood Spots for Human Immunodeficiency Virus Type 1 Drug Resistance Testing{triangledown}

Amanda McNulty,1 Cheryl Jennings,2 Diane Bennett,1 Joseph Fitzgibbon,3 James W. Bremer,2 Michael Ussery,3 Marcia L. Kalish,1 Walid Heneine,1 and J. Gerardo García-Lerma1*

Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia,1 Rush Medical College, Chicago, Illinois,2 Division of AIDS, NIAID, Bethesda, Maryland3

Received 29 September 2006/ Returned for modification 23 November 2006/ Accepted 3 December 2006

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at –20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at –20°C or at –70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that –20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.

* Corresponding author. Mailing address: Laboratory Branch, Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-4987. Fax: (404) 639-1174. E-mail: GGarcia-Lerma{at}cdc.gov.

{triangledown} Published ahead of print on 13 December 2006.

Journal of Clinical Microbiology, February 2007, p. 517-521, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.02016-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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