HIV DBS DNA PCR (Human Immunodeficiency Virus Dried Blood Spot Deoxyribonucleic Acid Polymerase Chain Reaction)
In Swaziland, approximately 15,000 children are living with HIV/AIDS, and there are approximately 54,000 AIDS orphans. Swaziland’s infant mortality rate, at 69 per 1000 live births, and the under-five mortality rate, at 120 per 1000 live births, have doubled over the past 15 years. HIV related mortality contributes significantly to both the infant mortality rate and the under-five mortality.
There are approximately 40,000 infant births each year, and with the antenatal HIV prevalence at approximately 40%, this results in 16,000 HIV-exposed infants per year in Swaziland. There has been a recent focus on the early diagnosis of HIV in infants and children less than eighteen months of age.
There are two different categories of tests used to diagnose HIV: antibody (indirect) tests and virologic (direct) tests. Antibody (indirect) tests include a rapid test, ELISA (Enzyme Linked ImmunoSorbant Assay), and Western Blot tests. Virologic (direct) tests include DNA PCR, RNA PCR (viral load), and P24 antigen testing. The tests available for early infant diagnosis in Swaziland are the rapid antibody test and the DNA PCR virologic test.
A diagnosis of HIV can be made as early as 4 weeks of age, using a polymerase chain reaction (PCR) assay that directly detects the presence of HIV DNA (not just the antibody). DNA PCR was introduced in Swaziland in February of 2007 at all of the 6 major hospitals (Mbabane Government Hospital, Raleigh Fitkin Memorial Hospital, Good Shepherd Hospital, Piggs Peak Hospital, Mankayane Hospital, Hlatikulu Hospital, and the Baylor Children’s Center of Excellence). The dried blood spot (DBS) technique is being used for DNA PCR testing, allowing for convenient blood collection and stable transportation of samples.
In areas where DNA PCR is not available, the rapid test that detects antibodies to HIV is commonly used. The rapid test poses difficulties in diagnosing HIV in children less than 18 months of age, because infants may still have maternal antibodies to HIV that cross over to the baby through the placenta during pregnancy. The maternal antibodies can persist in the infant’s blood for as long as 18 months. So, even if a child under 18 months is not infected, he may still have a positive rapid antibody test result, due to the presence of persistent maternal antibodies. While the rapid test can detect HIV exposure, it cannot be used to definitively diagnose HIV in infants and children less than 18 months.
Although the antibody test cannot definitively diagnose HIV infection in infants under 18 months of age, it can be useful for identifying potentially uninfected infants as early as 9 to 12 months of age, IF they have not breastfed or if they stopped breastfeeding at least 3 months before the antibody test. Virologic testing is necessary for a definitive diagnosis of HIV before 18 months.
DNA PCR is most accurate after 4 weeks of age (>98% sensitivity). According to Swazi protocols, all exposed infants should be tested for HIV with DBS DNA PCR at 6 weeks of age, or at the earliest clinical encounter before 18 months of age, regardless of their feeding practice. Unfortunately, the process of DNA PCR testing in the laboratory is labor-intensive and expensive, so rigorous quality control must be in place in order to receive accurate results.
HIV-infected infants are at increased risk for rapid disease progression and early death. Without treatment, over half of infected children will die before two years of age. Early diagnosis for HIV exposed infants using DNA PCR testing enables health care workers and mothers to initiate appropriate prevention, care, and treatment services to infants. Early treatment for infants and children improves their chances for survival, decreases infections, and increases their quality of life. Early HIV diagnosis can guide care and treatment and inform decisions on contrimoxazole prophylaxix, CD4 and other laboratory evaluation, and antiretroviral treatment. It can also assist health care workers in reinforcing important infant feeding recommendations.
PMTCT (Prevention of Mother to Child Transmission) programs have found that the antibody test used at 18 months was inadequate for clinical monitoring, due to high rates of mortality among HIV-positive infants, and lack of documentation of HIV exposure status after 18 months. Early diagnosis of HIV through DNA PCR testing at 6 weeks of age can give more accurate data on the impact of a PMTCT program.
That is, of course, if the test results are actually received at 6 weeks of age. Here’s the catch: DBS samples are being collected by trained health care workers at various facilities throughout Swaziland (including the BIPAI COE), and sent to the hospital that has been designated their “hub”. Each hospital then collects all of the samples received from facilities in their region, and transports them to the National Reference Laboratory (NRL) in Mbabane Government Hospital. Samples from throughout the entire country are then collected and sent to the testing site at the DNA PCR laboratory in Johannesburg, South Africa (NICD- National Institute for Communicable Diseases). The results then have to make their way all of the way back to the National Reference Laboratory in Mbabane Government Hospital, and then on to their respective Point of Care providers. This all takes, at the very least, six weeks, and costs $20 / test. By the time the mother and child have received their results, the child has been breastfeeding for another six weeks, and must receive yet another DNA PCR test in order to, again, confirm the absence of HIV in the baby. Realistically, no baby can be declared HIV FREE in Swaziland until 18 months of age.
What will it take to bring infant HIV testing to Mbabane, Swaziland? Here are a few ideas that I have come across (by far, my favorite is #3):
1. An experiment done in Cambodia by Janin Nouhin and Marie Nguyen showed that “boosted-p24-antigen profile assay, with performances similar to viral cultures and costs similar to DNA-PCR, is easier to perform and could readily be set up in resource-poor settings.” Although the p24 antigen test is NOT recommended for infant testing, due to its low sensitivity in babies, these results show that perhaps a boosted-p24-antigen profile assay could be the answer.
Source: http://www.ajtmh.org/cgi/content/full/75/6/1103
2. A study done in Argentina showed that centrifugation improves the detection of HIV p24 antigen in the plasma of infants born to mother infected with HIV. The aim of the study was to improve the sensitivity of the detection of p24 by centrifuging the plasma. The results showed that “centrifugation of plasma samples prior to determination of p24 in pediatric patients resulted in a significant increase in sensitivity.”
3. Pocket-size PCR Machine: This machine is “a novel thermocycling system capable of performing high-speed DNA amplification via the PCR in a simplified, inexpensive and portable format. The advantageous features of this technology include an inexpensive hardware platform which can be build for approximately $10 (compared to the estimated cost of $2500 in 2004), timescales of the order of 10-20 minutes (presently over 1 hour), no moving parts, no external fluid transport beyond sample loading and unloading, requires little or no modification to existing reaction protocols, portability and small size (using 2 AA batteries). It is ideally suitable for performing PCR based assays in situations where a yes-no result is desired and an extensive laboratory infrastructure is lacking.”
*Seems too good to be true, right? I’ve emailed the professor at Texas A&M in hopes of obtaining more information.
Sources:
http://otc.tamu.edu/technologies.jsp?casecode=2418TEES06
http://www.rsc.org/chemistryworld/News/2007/May/01050701.asp
If anyone knows of other solutions to this issue, please leave a comment. Lesotho guys, what is the DNA PCR status there? As of now—we have identified the turnaround time of HIV DNA PCR results as a substantial concern at the Baylor clinic in Swaziland, and are considering several existing solutions. Perhaps this would be a decent project for students at Rice. What will it take to bring quick, inexpensive, simple HIV testing to newborn infants in developing countries? Perhaps this idea is a little ambitious. Perhaps I still have quite a bit to learn about DNA PCR.