Simple DNA Extraction Method for Dried Blood Spots and Comparison of Two PCR Assays for Diagnosis of Vertical Human Immunodeficiency Virus Type 1 Transmission in Rwanda

Abstract

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the pol region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

“…In the last 25 years, DBS have been used for various purposes, including detection of HIV antibodies (2, 5, 18, 23, 31), detection of HIV antigen (22,32), early diagnosis of perinatally infected infants (4,15,28,30,35), quantification of CD4 ϩ T cells (26), and quantification of viral plasma RNA (1,3,7,9,16,25,29). Few studies examined the possibilities of performing drug resistance testing on dried blood, plasma, or serum on filter paper (24,33,43).…”

“…The procedure to extract DNA from the DBS was adopted from that of Fisher et al (15). Briefly, one whole DBS was cut into small pieces and transferred to a 2-ml tube.…”

The use of dried blood spots on filter paper for the diagnosis of HIV-1 in infants born to HIV seropositive women

“…In a previous study, using Isocode cards, the sensitivity and speciÞ city of the PCR using DBS were 90 and >98%, respectively. [6] According to the National AIDS Control Organization in India, with approximately 27 million pregnancies each year and an overall estimated 0.3% prevalence rate of HIV infection among pregnant women, it is estimated that around 100,000 HIV-infected women deliver every year. [7] Early diagnosis of HIV-1 infection in infants cannot be accomplished with conventional antibody tests due to the persistence of passively transferred maternal antibodies for up to 18 months after birth.…”

Molecular Surveillance as Monitoring Tool for Drug-Resistant Plasmodium falciparum in Suriname

“…Genomic DNA of all samples was extracted from DBSs with a slightly modified Chelex method. 21 pfcrtK76T determination. Analysis was performed on all samples (N = 80).…”