Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 26, Num. 1, 2008, pp. 71-74

Indian Journal of Medical Microbiology, Vol. 26, No. 1, January-March, 2008, pp. 71-74

Brief Communication

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

Jacob SMini, Anitha D, Vishwanath R, Parameshwari S, Samuel NM

Department of Experimental Medicine, The Tamilnadu Dr. MGR Medical University, Guindy, Chennai-32, Tamil Nadu
Correspondence Address:Department of Experimental Medicine, The Tamilnadu Dr. MGR Medical University, Guindy, Chennai-32, Tamil Nadu, nms_mds@yahoo.com

Date of Submission: 11-Sep-2006
Date of Acceptance: 23-Mar-2007

Code Number: mb08016

Abstract

Keywords: Dried blood spots, HIV-1 DNA PCR, HIV infection in infants, MTCT

HIV-1 transmission from an infected mother to her infant is estimated to be 21-43% in less-developed countries. ^[1] Simple and inexpensive assays are necessary for the diagnosis of HIV-1 infection in infants. However, early diagnosis of HIV-1 infection in infants cannot be accomplished with conventional antibody tests due to maternal antibodies for up to 18 months after birth. ^[2]

Polymerase chain reaction (PCR) is the preferred method to determine the HIV-1 infection status in infants born to HIV-1 seropositive women. PCR is commercially available and has high sensitivity and specificity. An independently validated qualitative HIV-1 DNA nested PCR of gag gene has been used on specimens from infants. Advantages of this assay include the ability to adapt assay reagents to local reagents, the potential to automate for large-scale testing and the ability to produce the results within a day. ^[3]

Blood spotted onto filter paper facilitate the collection, transport and storage of blood samples for laboratory use. In 1963, Guthrie first published data demonstrating the feasibility of collecting neonatal blood samples onto filter paper for phenylketonuria testing of newborns. ^[4] In 1987, Edward McCabe reported successful extraction of DNA from dried blood spots (DBS) collected on filter paper. ^[5] Once dried, the blood specimens on filter paper are no longer infectious.

The objective of this study was to evaluate the use of DBS specimens for PCR and to compare the specimens with whole-blood stored in tubes for HIV-1 DNA PCR.

Materials and Methods

Between January 2005 and May 2006, 577 infant blood samples in EDTA collection tubes were received in our department from various institutions in South India. Samples were collected from infants at 48 hours of birth (n = 359) and second month after birth (n = 218). These samples were stored as whole-blood at -20 °C until assayed. An HIV-1 qualitative in-house nested DNA PCR was performed from whole-blood for all infant samples. Positive samples tested from whole-blood along with every fifth negative sample were coated onto the filter paper.

Specimens were thawed by placing at room temperature and mixed by pipetting; then DNA extraction was performed from the whole-blood using QIAamp ^® DNA blood mini kit (Catalog no. 51104; Qiagen GmbH, Germany). According to the kit protocol, 200 μL of whole-blood was used for DNA extraction and the extracted DNA was stored at -20 °C for the PCR amplification.

Aliquots of the thawed whole-blood were spotted onto Isocode Stix (Schleicher and Schuell, Keene, NH, USA). Each Isocode Stix [Figure - 1] contains four triangular sample collection areas, perforated for easy removal of the device. Ten microlitres of whole-blood was applied onto each triangle. The filter paper was then allowed to dry for a minimum of three hours. When thoroughly dry, each Stix was placed in a zip lock storage bag with desiccant, stored at room temperature and protected from light. All the four triangles (matrix) were used for template isolation. Each triangle was placed over an open sterile 2 mL microcentrifuge tube and while closing the cover the end of the stick was pulled gently until the triangles detached and fell into the tube. The triangles were washed with 500 μL of sterile water by pulse vortexing for 5 seconds. The matrix was removed using sterile fine-point forceps squeezing along the side of the tube to remove excess water. The matrix was then placed into a 0.2 mL sterile microcentrifuge tube and 100 μL of sterile water was added. This was transferred onto a heating block or boiling water bath at 95-100 °C for 15-30 min. At the end of incubation, the sample was pulse vortexed 60 times and briefly centrifuged. The matrix was removed by squeezing along the side of the tube to remove excess water. The 100 μL elute contains DNA template for amplification.

The independently validated nested PCR was used: 10 μL of DNA solution extracted from whole-blood and from DBS was used for PCR amplification (using gag primers) in a 100 μL reaction mixture. Master mix for first round PCR was 10x buffer: 10 μL; dNTPs (25 mm): 1 μL; Taq DNA polymerase (2 units/μL): 1 μL; primer 1 sequence (GAG 1): TCT CTC GAC GCA GGA CTC GGC TTG CTG (25 pmol): 0.5 μL; primer 2 sequence (GAG 2): TAA CAT TTG CAT GGC TGC TTG ATG TCC (25 pmol): 0.5 μL; and water: 77 μL.

Ninety microlitres of master mix was added to 10 mL of DNA. The reaction conditions for the first program was to hold at 94 °C for 1 minute, 55 °C for 1 minute, 72 °C for 1 minute, 32 cycles at 94 °C for 15 seconds, 55 °C for 45 seconds, 72 °C for 1 minute, extension at 72 °C for 5 minute and at 4 °C for infinity. The master mix composition for second round PCR was same as first round except the water volume and the primers as follows - primer 3 sequence (GAG3): CTA GAA GGA GAG AGA GAT GGG TGC GAG (25 pmol); primer 4 sequence (GAG 4): CTT GTG GGG TGG CTC CTT CTG ATA ATG (25 pmol); water: 82 μL.

Five microlitres of first round PCR product was used in the 95 mL master mix composition. The time program was same as first round PCR except the annealing temperature, which was changed from 55 to 65 °C to increase the specificity. One confirmed positive sample and one confirmed negative control were included in every assay.

To determine if the PCR is positive, 5 μL from each nested PCR was loaded onto an 1% agarose gel along with DNA ladder (1 kb). After electrophoresis, the gel was stained with ethidium bromide. The PCR products at 650 base pairs were visualized using a UV transilluminator.

Results

Qualitative HIV-1 DNA PCR using whole-blood frozen in tubes

During the study period, 577 infant samples were tested for HIV-1 infection by the qualitative in-house nested DNA PCR. A total of 359 (62%) samples were collected from infants at 48 hours of birth and 218 at second month after birth (38%). At 48 hours of birth, 19 of the 359 (5.29%) samples were HIV-1 positive and 340 (94.7%) samples were negative for HIV-1 DNA.

At second month after birth, 19 (8.7%) of the 218 samples were positive and 199 (91.2%) were negative [Table - 1].

Qualitative HIV-1 DNA PCR using dried blood spots

All of the specimens testing positive from the whole-blood samples and every fifth negative sample were coated on the filter paper. DNA was extracted from the DBS on filter paper and was amplified using in-house nested PCR.

Of 19 positive samples, 18 (95%) tested positive and 1 tested negative. The 68 specimens that tested negative when whole-blood was assayed were also negative in the DBS test. Therefore, the sensitivity of the test is 95% and the specificity 100%.

At second month after birth, 19 were positive and 40 negative samples (every fifth sample of 199) were negative [Table - 2]. Therefore, the sensitivity and specificity was 100% [Table - 3].

Discussion

In the present study, we have performed in-house qualitative HIV-1 DNA PCR for infant samples (at birth and at second month after birth). HIV-1 DNA PCR was performed for whole-blood samples and for DBS coated on filter paper. This study performed a total of 722 DNA PCR tests. Nineteen infant samples were found to be HIV-1 positive using whole-blood and 18 of these were positive using DBS. Three hundred and forty infant samples were HIV-1 negative using whole-blood. Every fifth negative sample was coated on the filter paper; therefore, 68 negative samples were tested using HIV-1 DNA PCR and the results were concordant.

However in the second month, the results were concordant for 19 positive infant samples tested by PCR using whole-blood and DBS. Two hundred and eighteen infant samples were negative using whole-blood and 40 samples (every fifth sample of 218) coated on the filter paper were negative.

HIV-1 DNA PCR assay using DBS offers a sensitive and specific test appropriate for the diagnosis of HIV-1 in infants. In this study, the sensitivity and specificity of HIV-1 DNA PCR using DBS is 95 and 100% in birth samples. In a previous study, using Isocode cards, the sensitivity and specificity 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. ^[8]

Detection of HIV-1 DNA by PCR is an established method for determining infection status in children born to HIV-1-seropositive mothers. ^[9] PCR testing at birth is to obtain the earliest possible identification of infants infected in utero; and at two months is to identify all perinatally infected infants. DNA PCR sensitivity increases dramatically by two weeks and reaches 96% by one month of age. ^[10] Two positive HIV-1 DNA PCRs at any time confirms HIV-1 infection. Once infection is confirmed, further HIV-1 DNA PCR testing is not required.

There are many advantages of the use of HIV-1 DNA PCR with dried blood specimens over HIV-1 DNA PCR with whole-blood specimens. ^[11] Whole-blood can easily be coated on the filter paper from heel stick or finger punctures in infants; thus avoiding the use of syringes and vaccutainer tubes. Blood coated on filter paper lyses the cells and binds the DNA. Therefore, the sample centrifugation and extraction procedures are reduced. Dried blood on filter paper appears biologically stable ^[12] and can be stored at room temperature. It can be transported easily and therefore it is convenient to use the DBS in resource limited settings.

In conclusion, qualitative in-house nested PCR using DNA extracted from filter paper permits the diagnosis of HIV-1 infection among infants born to HIV-1 seropositive mothers. This assay is simple, rapid, sensitive and specific and can be used in resource limited settings.

Further studies need to be evaluated using the filter paper directly coated with whole-blood from finger or heel stick in infants.

References

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