Pathfinder International works to improve the reproductive health

of women, men, and adolescents throughout the developing world.

Without treatment, an infant infected with HIV in

Africa has a 35 percent chance of dying by his first

birthday and a 53 percent chance of dying before

the age of two.� But if the baby receives prophylactic

antibiotics, such as cotrimoxazole, soon after birth and

Antiretroviral Therapy (ART) as soon as is medically

indicated, he has a good chance of surviving childhood

and living a long, healthy life.

The challenge in resource-limited settings is identifying HIV-infected infants and providing early access to this life-saving medicine. Access to Antiretroviral (ARV) drugs has improved in Kenya in the last few years, spurred in large part by the introduction of the President’s Emergency Plan for AIDS Relief. But until very recently, little could be done to diagnose infants’ HIV status in their first year of life.

The standard methods of diagnosing HIV infection in adults—enzyme linked immunoassay and Western blot immunoassay—test for antibodies to the virus. Because mothers pass their antibodies to their babies while still in the womb, the standard assays cannot accurately diagnose infants until the age of 18 months, the earliest age at which the mother’s antibodies are no longer present in the infant’s blood. By this time, many HIV-infected infants will have died.

The test used to diagnose babies born to HIV-infected mothers in developed countries, Polymerase Chain Reaction (PCR), tests directly for HIV DNA rather than the HIV antibody. It requires sophisticated, expensive equipment not available in rural settings. Traditionally, the test requires a liquid blood sample, which if taken in a rural area and transported to a testing facility, needs to be kept refrigerated.

1 Newell, M.L. et al. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. The Lancet, Volume 364, Issue 9441, 2 October 2004-8 October 2004, Pages 1236-1243 (http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1B-4DFJD85-15&_user=10&_coverDate=10%2F08%2F2004&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=209800a5547081904f7316ab8e150e59) (7/23/07)

Recently, however, a new technology has emerged that allows PCR to be performed on small spots of dried blood. The Dried Blood Spots (DBS) are easy to prepare in a resource-limited setting and can be stored and shipped to testing facilities without refrigeration. Infants can be tested using PCR as early as six weeks of age. PCR testing using DBS has been proven to be as effective as PCR using liquid blood samples, with sensitivity (percentage of results that will be positive when HIV is present) of 100 percent, and a specificity (percentage of results that will be negative when HIV is not present) of 99.6 percent.2

Pathfinder international’s resPonse

With funding from the Centers for Disease Control and Prevention, Pathfinder International/Kenya is

2 Sherman, Gayle G MD, Dried Blood Spots Improve Access to HIV Diagnosis and Care for Infants in Low-Resource Settings. JAIDS Journal of Acquired Immune Deficiency Syndromes. 38(5):615-617, April 15, 2005. (http://www.jaids.com/pt/re/jaids/abstract.00126334-200504150-00016.htm;jsessionid=GVFfnbLpy4JRzg2sTKVWvHXKQ7J8BBp5pfhpv1JSjMZZT6LS0S12!-1740698184!181195629!8091!-1) (7/11/07)

Early Infant Diagnosis of HIV through Dried Blood Spot Testing: Pathfinder International/Kenya’s Prevention of Mother to Child Transmission Project

OCTOBER 2007

DBS testing allows for diagnosis of HIV as early as six weeks of age. Babies have the best chance of thriving when they are diagnosed early in life and recieve proper health care and nutrition.

Photo: Pathfinder International/Kenya PM

TC

T team

implementing a Prevention of Mother-To-Child Transmission (PMTCT) project in 14 districts of Kenya, covering a population of 5.4 million. In 2006 there were 10,000 infants born to HIV-infected mothers in the project areas, but only 300 were seen and tested for HIV at the standard age of 18 months; 47 percent tested positive for HIV. This transmission rate, however, cannot be seen as indicative of the larger group’s transmission rate. The small sample that was tested was a biased group; most of the children who were brought in at 18 months for testing were sick. Parents of healthy children often do not see the need to have them tested. But the figures do illustrate the difficulty in following HIV-infected mothers and their children over long periods of time and in ensuring appropriate care and treatment when no diagnosis has been made. There are many reasons babies are lost to follow-up. Many die; others are orphaned and have no one to bring them back to the clinic for care. In the slums and poor areas of Nairobi, which account for a large portion of the project’s target population, communities are fluid, creating additional barriers to follow-up.

In an effort to identify HIV-infected babies earlier, Pathfinder/Kenya began integrating DBS testing into its PMTCT program in August of 2006. Since then, Pathfinder has trained 93 nurses and lab technicians in 31 of the PMTCT project-supported clinics. The project plans to provide DBS training and support to 30 more clinics by November 2007 and to all 230 project-supported clinics by the project’s end in 2010.

Over the first three months of implementation, 546 samples were collected in project-supported clinics and transported to the CDC-KEMRI laboratory in Nairobi for testing.3 Only four of these samples were rejected by the lab as being of insufficient quality for testing—this is a remarkably low percentage compared to results from other programs. Of the 542 samples on which PCR was performed, 90 (16.6 percent) tested positive for HIV.

All infants who tested positive were referred to centers providing pediatric ART, where they undergo further evaluation on the need for Highly Active Antiretroviral Therapy (HAART). Those who meet the national guidelines for treatment are started on HAART. HIV- infected infants not yet in need of treatment are given prophylactic cotrimoxazole and nutritional support.

3 Laboratories with DBS testing capability are still extremely limited in Kenya. The CDC-KEMRI lab is the lab closest to project sites that is able to perform the test.

PATHfInDER InTERnATIOnAl

Choosing the location to draw blood is important in preparing a DBS sample. If the area does not bleed sufficiently, the sample may be too small to test. The following table helps providers decide where to draw blood for a DBS sample.

age, weight Where to draw blood

�-4 months, less than 6 kg Heel

5-�0 months, less than �0 kg Toe

larger than �0 kg finger

The number of babies brought to the project clinics for testing during the three-month period for which data is available (546) is 183 percent greater than the number tested at these clinics in the entirety of the previous year (299). When mothers know there are medicines and services available for their infants if they test positive for HIV, they are more likely to have their children tested.

Parents are counseled before the test to ensure their understanding of the procedure and the resources available for them if their child tests positive. Just as importantly, parents are counseled for the possibility of a negative test. Because HIV can be transmitted through breast milk, the mothers of babies who test negative and are breastfeeding often wish to stop breastfeeding immediately, regardless of their ability to safely provide replacement feeding. They are therefore counseled before testing on the risks and benefits of both continuing to breastfeed or of introducing replacement feeding. Clinic staff support parents in assessing the acceptability, feasibility, affordability, sustainability, and safety of replacement feeding by analyzing the family’s access to clean water, electricity, hygienic latrines or toilets, their financial situation, and other social and economic factors necessary to support replacement feeding. If they are unable to sustain safe replacement feeding and choose to continue breastfeeding, the babies should be tested again at 18 months of age.

dBs ColleCtion ProCedure

Pathfinder developed a simple two-day training program that teaches clinic staff how to prepare and store the blood samples. The steps included in sample collection are:

Warm the baby’s foot (or hand, if older than 10 months or larger than 10 kilograms) to facilitate blood flow. This can be done by wrapping a hand around the foot while the baby sits in its mother’s lap.

Position the baby with its feet down.

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overall sample size and should be looked at again once more data has been collected. The high rate of transmission seen in caesarian deliveries and the low rate of transmission seen in mixed feeding are also worth investigating, though both of these results could be explained by a small sample size (only 4 caesarian births were reported and only 10 infants received both breast milk and other food.) It is important to note that the caesarian births were performed for complicated labors, which could have disrupted the usual placental barrier causing the infant to be exposed to the mother’s blood during labor. None of the caesarian births were planned for the purpose of PMTCT.

Early Infant Diagnosis of HIV through Dried Blood Spot Testing

Above is an example of a collected blood sample.

Clean the baby’s foot with disinfectant and let it dry.

While wearing gloves free of powder, the provider pricks the baby’s foot with a lancet to draw blood.

The first drop of blood should be wiped away with gauze or cotton wool.

The provider should then allow a large drop of blood to collect on the foot before touching it to the circle on the filter paper. The circle should be filled completely by the blood drop and at least two circles should be filled per card. (See photo above.)

The foot should then be cleaned with disinfectant and left unbandaged.

Samples should be stored horizontally out of direct sunlight for at least three hours. Once dry, samples are stored in sealable plastic bags with desiccant packets and a humidity card and are ready for transport to the laboratory. If not sent that day, samples should be refrigerated—though they need not be refrigerated during transport.

analysis of Preliminary results

The results of 182 samples were analyzed according to the infant’s age at testing, sex, type of feeding, history of ARV prophylaxis, type of delivery, and whether or not the mother received ARV prophylaxis during labor. As expected, fewer infants who received ARV prophylaxis tested positive than those who didn’t, and fewer infants born to mothers who received ARV prophylaxis tested positive than those born to mothers who did not receive ARVs. Surprisingly, more than 23 percent of males tested positive, while only 11 percent of female infants tested positive. This result might be attributable to the small

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Percent hiV positive

Percent hiV negative

age (weeks)

<6 24 76

>6 – 25 �8 82

>25 - 52 �3 87

>52-77 0 �00

sex

female ��.5 88.5

Male 23.4 76.6

type of feeding

Breast 20 80

*Mixed 20 80

Replacement �5 85

Prophylaxis

Infant received ARVs

�5 85

Infant did not receive ARVs

30 70

Mother received ARVs

�6 84

Mother did not receive ARVs

20 80

type of delivery

*Caesarian 50 50

Vaginal �6 84

*denotes small sample size

table 1. Results of DBS Testing on �82 Infants in the Project Area

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9 Galen Street, Suite 2�7 • Watertown, MA 02472 USA • Tel: 6�7-924-7200 • fax: 6�7-924-3833 • www.pathfind.org

1007/4K

ConClusion

The Pathfinder program has shown excellent results thus far. Its main hurdle has been overcoming transportation barriers to reduce the time lag between data collection and client receipt of the results. Timely delivery of results is necessary to ensure clients return to the health facility after testing. Though the ideal turn-around time of two weeks between sample collection and delivery of results has not yet been reached, it has improved from an average of four weeks to three. Expanded laboratory capacities and additional resources for health facilities to ensure sample transport would do much to improve the situation.

Counseling will continue to be an important part of early infant diagnosis. Though knowing an infant’s sero-status can help parents provide the care their infant needs, it can also create confusion. There are reports of some parents neglecting their HIV-infected infants out of fear and misunderstanding. Some mothers assume that because their infant has tested negative, their future babies will be free of the disease as well. The choice of whether or not to continue breastfeeding an HIV-negative baby is difficult—continuing to breastfeed means continuing to put the child at risk of infection, but in many areas replacement feeding represents an even greater risk to the infant’s health and survival. Good counseling and ongoing care and support are vital to helping parents make informed decisions that are right for their family.

The introduction of early infant diagnosis techniques

is an important step in the fight against HIV/AIDS in

Africa. By identifying infants who need services as

early as possible, these children will have a better

chance of maturing into healthy adults. Widespread

use of early infant diagnosis programs will help

funding agencies and program implementers analyze

the results of PMTCT programs and illustrate the need

for increased funding for ART.

A provider prepares to collect a DBS sample. Keeping the foot warm and pointed down helps facilitate blood flow and makes sample collection easier.

Photo: Dom

inic Karanja, Pathfinder International/K

enya