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Early Diagnosis of HIV Infection in HIV-Exposed Infants: Can Complexity and Cost Be Overcome in Resource-Poor Settings?

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Introduction

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Challenges of Diagnosing HIV Infection in Infants

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Most HIV infection in children results from mother-to-child transmission (MTCT) of HIV, which can occur during pregnancy, labor and delivery, or the breast-feeding period. Despite the great advances that have been made in developing and implementing effective interventions to prevent HIV transmission from infected mothers to their infants, almost 2,000 infants in resource-poor countries are infected with HIV every day through MTCT.( 1 ) As of 2006, there were approximately 2.3 million HIV-infected children worldwide.( 2 ) That number is expected to increase steadily in the near future for several reasons. Currently, less than 10% of HIV-infected pregnant women in resource-poor countries receive antiretroviral prophylaxis for the prevention of mother-to-child transmission (PMTCT).( 3 ) Even if antiretroviral prophylaxis services were scaled up dramatically, HIV infection in children would continue to increase unless there were a concurrent increase in services to prevent new HIV infections in women, improve access to family planning, and expand the availability of antiretroviral treatment (ART) for women who need it.

As with adults, HIV-infected children respond well to ART. However, such treatment is most effective when it is initiated before the child becomes ill (ie, before advanced disease progression). Without ART, HIV infection progresses very rapidly in infants and children. In resource-poor settings, approximately 30% of untreated HIV-infected children die before their first birthday and more than 50% die before they reach 2 years of age.( 4 ) Untreated HIV infection in children also may result in growth delays and mental retardation that may not be reversed by ART. It is therefore crucial to diagnose HIV-exposed infants as early as possible in order to prevent death, illness, and growth and developmental delays.

Because of their low cost, simplicity of use, and ability to provide quick results, rapid antibody tests are the most commonly used assay for diagnosing HIV infection in resource-poor countries. However, because HIV antibodies cross the placenta during pregnancy, all infants born to HIV-infected mothers will have received maternal antibodies in utero and will test antibody positive at birth regardless of their own HIV infection status. Maternal antibodies do not completely dissipate until 12-18 months after birth, and all antibody tests for HIV-exposed infants taken before that time are therefore unreliable.( 5 , 6 )

Another difficulty in diagnosing pediatric HIV infection in infants in resource-poor countries is ongoing exposure to HIV in breast-fed infants, which makes it difficult to exclude HIV infection until after breast-feeding has stopped completely.

Because of these complications, most infant HIV testing in resource-poor countries is performed using rapid antibody tests at 18 months of age. However, by this age, many infected babies will have died and many more will have been lost to follow-up. An inexpensive, easy-to-use HIV test that would be accurate for HIV-exposed infants less than 18 months of age is desperately needed. Such a test could prevent millions of premature HIV-related deaths.

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Clinical Approaches to Diagnosis

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Clinicians in resource-poor countries who lack the ability to definitively diagnose HIV-infected infants less than 18 months of age can use clinical approaches such as those outlined in the Integrated Management of Childhood Illnesses (IMCI) algorithm to guide their care and treatment decisions.( 7 , 8 ) The algorithm trains health care workers to recognize common signs of HIV infection in children such as parotid swelling or pneumonia. The usefulness of these algorithms is debatable, particularly as HIV-infected infants may be asymptomatic for extended periods of time. In a "letter to the editor" critiquing the IMCI algorithm, a group of South African clinicians discussed their experience in retrospectively applying South Africa's IMCI guidelines to infants in a PMTCT study.( 9 ) They found that only 17% of infected infants would have been diagnosed at 6 weeks of age, and only 50% at 12 months of age, significantly lower than the 70% rate cited in an earlier study promoting use of the algorithm.( 10 ) The "fallback" position for diagnosis is to wait until infants exhibit signs of advanced HIV infection, but ART is more likely to fail when initiated for patients with advanced HIV disease than it is when initiated for asymptomatic patients.

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The Ideal Test

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The ideal diagnostic test for HIV-exposed infants would meet the following criteria:

transparent gif bullet It would have a specificity of >99% for all HIV subtypes, and a sensitivity of >98%.
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transparent gif bullet Health care workers would be able to perform the tests on dried blood spots (DBS) placed on filter paper.
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transparent gif bullet The test would use economical, simple, and low-maintenance technology, with readily available replacement parts and equipment.
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transparent gif bullet The test would use inexpensive reagents that would not require refrigeration.
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transparent gif bullet The test methodology would be easy to teach and would be able to be performed without extensive training.
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transparent gif bullet The test would cost less than $2 to administer.
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transparent gif bullet The test results would not be influenced by maternal ART for HIV infection during pregnancy or antiretroviral prophylaxis for the mother or infant to prevent MTCT.
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Currently, no single test for the diagnosis of HIV infection meets all these criteria.

The sensitivity of a test is the probability of its giving a positive result if infection is truly present. As the sensitivity of the test increases, the proportion of false-negative results decreases.

The specificity of a test is the probability of its giving a negative result if infection is truly absent. As the specificity of the test increases, the proportion of false-positive results decreases.

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Potential Benefits of Early Diagnosis: A Caution

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How beneficial would an "ideal" test for HIV infection of infants be? In a recent article in Nature, Aledort et al discuss the potential health benefits from a hypothetical new test that would accurately diagnose HIV infection in HIV-exposed infants less than 12 months of age.( 11 ) The researchers calculated the health care benefits that would occur with the test (at varying levels of performance) and compared them to the health outcomes under current conditions, in which there is usually no HIV testing available for infants under the age of 12 months. The outcomes measured were annual health benefits in terms of life years saved, disability-adjusted life years (DALYs) saved, and the proportion of total disease burden averted. The researchers based their analysis on the current figures for HIV test performance and pediatric ART availability and assumed that, in the absence of an appropriate HIV test, asymptomatic infants would be evaluated using IMCI diagnosis algorithms. They also assumed that symptomatic infants diagnosed by the IMCI algorithm would be classified under the World Health Organization (WHO) clinical stage 3 or 4.

The researchers found that even an ideal test would have little impact on adjusted life years saved without an increase in the availability of ART for children. Currently, only 1 in 10 children in need of ART receive it.( 12 ) The authors further conclude that, as long as the initiation of ART in children is determined primarily by clinical staging, which excludes infants without symptoms or access to health care workers trained in the ICMI algorithms, the potential for a new HIV diagnostic test to reduce disease burden is limited.

These caveats are important for framing discussions about HIV diagnosis in infants. The development of new diagnostic tests must coincide with an improvement in the recruitment, training, and retention of health care workers in reproductive and child services and with an increase in the availability of low-cost, fixed-dose combination antiretroviral drugs in pediatric formulations.

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Antibody Testing for Pediatric HIV Diagnosis

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As mentioned above, making definitive diagnoses of HIV infection for infants exposed to maternal HIV is difficult because of the presence of maternal HIV antibodies, which are passively transferred across the placenta to the infant.( 13 , 14 ) This process involves only antibodies of the immunoglobulin G (IgG) class under normal conditions, providing passive protection for infants against a variety of infectious agents for as long as 18 months.( 15 ) After maternal antibodies dissipate, antibodies to microbial agents represent the immunologic response of the infant.

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IgM Antibody Tests

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In most instances of in utero infection, the antibody consists of both an IgG and an immunoglobulin M (IgM) antibody response.( 16 ) Because IgM is a high molecular weight antibody, its presence in the infant's circulation represents an immunologic response of the infant to infection. Thus, the presence of IgM antibody response to cytomegalovirus, toxoplasmic organisms, or other microbial agents that can cause infection of both mothers and infants is indicative of infection in the infant.( 17 )

When the U.S. Food and Drug Administration licensed the enzyme-linked immunosorbent assay (ELISA) in 1984, there was hope that it could be used for early diagnosis of HIV infection in HIV-exposed infants.( 18 ) However, the ELISA used a methodology that measured only IgG antibody and therefore could not differentiate passively transferred maternal IgG antibodies from IgG antibodies produced by an infected infant. Based on previous observations that a diagnosis of congenital infections could be made by testing for IgM antibodies, modifications of the ELISA were made to detect IgM antibody response to HIV. For reasons that are not clear, neither infants infected with HIV in utero nor those infected intra- or postpartum respond with production of IgM antibodies. This precludes using an IgM antibody test as a means of differentiating passively transferred maternal antibodies from those actively produced by the infant.

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IgA Antibody Tests

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A different approach for early detection of HIV in exposed infants was to test for evidence of immunoglobulin A (IgA) antibody responses to HIV infection. IgA, as with IgM, is not transferred across the placenta in significant amounts. A study using a technique called Eli-spot assay found a small percentage of infants with the IgA antibody who were later shown to be infected.( 19 ) However, because of the insensitivity of the Eli-spot methodology and its complexity, clinicians and researchers did not adopt it for infant diagnosis.

In the early 1990s, Landesman and associates used another approach to investigate IgA antibodies.( 20 ) They used the simple measurement of IgA antibodies in a modified IgA antibody capture ELISA in serial plasma samples obtained from HIV-exposed infants. Initially, all samples were positive. By 8 months of age, all HIV-uninfected infants were found to be antibody negative. Although the IgA HIV antibody disappeared more rapidly than the IgG antibody did in IgG-based assays, the detection of IgA antibodies was still not useful for diagnosing infection in young infants (ie, those less than 8 months old). In addition, the cost of the tests and equipment in comparison with the rapid antibody approach would be prohibitively higher in resource-poor countries.

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New Uses for Antibody Tests

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Although current guidelines do not recommend using antibody tests to diagnose HIV infection in infants less than 18 months of age, there is growing recognition that these tests can play an important role in identifying potentially infected infants who might require further virological testing. Draft WHO diagnostic guidelines recommend the use of antibody tests in infants less than 18 months of age for:

transparent gif bullet Identifying children who are HIV exposed when the maternal HIV status is not known; identifying these children would allow them to obtain follow-up virological testing and appropriate care such as cotrimoxazole prophylaxis
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transparent gif bullet Supporting a presumptive clinical diagnosis of severe HIV disease in children to allow initiation of ART
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transparent gif bullet Excluding HIV infection in HIV antibody-negative, HIV-exposed children less than 18 months of age who have never breast-fed or who have discontinued breast-feeding for more than 6 weeks ( 21 )
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Culture of HIV from Blood

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Early in the HIV epidemic, HIV blood culture was use to detect HIV infection and to measure the amount of virus in blood directly.( 22 , 23 , 24 ) HIV culture also was used for infant diagnoses and as a means of determining the severity of infection and subsequent response to treatment in both adults and children. Although the test was sensitive and specific, and it could be used to quantify a patient's viral load, this method has never been used on a large scale for diagnosis because the testing techniques are complex and require costly reagents and equipment, prolonged laboratory time, and large volumes of blood. In addition, up to 7 days may be needed to obtain results and, because the virus culture contains viable HIV, specialized biohazard equipment is required.

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P24 Antigen Assays

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Before the development of HIV RNA and DNA polymerase chain reaction (PCR) techniques for the diagnosis of HIV infection and measurement of viral load, HIV p24 antigen assays were used to measure viral load. HIV p24 is a protein product of HIV replication that is present in varying amounts in the blood of HIV-infected individuals. Because HIV p24 is an immunogenic protein, HIV-infected individuals also form antibodies to p24. Thus, it is present in the blood in the form of free p24 and antibody-bound p24 (immune complex). In order to measure total p24 antigen, it is necessary to disassociate antibody from antigen. Techniques have been developed to perform this task, but have not been entirely successful. Nevertheless, because of the simplicity of the assay and its relatively low cost, researchers have pursued improvements in the assay performance even though HIV RNA PCR assays are more sensitive and specific.

This research has been partially successful. Laboratory techniques have been developed to disassociate p24 immune complexes, improving the test's quantitative ability and, in the case of HIV-exposed infants, diagnostic performance.

Several studies have found that ultrasensitive p24 antigen tests can reliably detect HIV infection in infants older than 6 weeks of age with a specificity and sensitivity similar to that of HIV DNA PCR and HIV RNA assays.( 25 , 26 , 27 , 28 ) The tests are accurate on many HIV subtypes and are easier to perform than other virological assays. Their cost is similar to the cost of the later-generation PCR tests.

Concerns about the sensitivity of p24 tests remain. In a recent study of these new "ultrasensitive" quantitative HIV p24 antigen assays, Knuchel et al compared the sensitivity of the tests when used on DBS and on plasma.( 29 ) They found that the tests had 100% specificity with no difference in quantitative results between DBS and plasma. They also compared their p24 antigen results with HIV RNA and found a positive correlation, but the correlation coefficient was low (r = 0.67). The sensitivity of the HIV p24 assay compared with that of the HIV RNA test was about 90%. This would mean that using the assay to screen HIV-exposed infants would result in as many as 10% of the infants being misdiagnosed as uninfected. A more recent comparison of testing methodologies conducted by Knuchel et al with 72 pediatric specimens from Tanzania and 210 pediatric or adult specimens from Switzerland found even lower sensitivity. The detection rates for the different assays were 84% for p24 antigen tests on DBS; 79% for DNA PCR tests conducted on DBS; 85% for p24 antigen tests on plasma; and 100% for RNA PCR tests performed on plasma.( 30 )

Even with significant improvement in the efficiency, economy, and specificity of HIV p24 antigen assays, the sensitivity of these tests will remain problematic. The cost savings that they offer may not offset the clinical implications of failing to diagnose a significant percentage of HIV-infected infants.

More important than concerns about test sensitivity is the fact that the equipment and consumables required for p24 antigen tests currently are not available for large-scale programs. In addition, there are theoretical concerns that the p24 antigen assays may have reduced sensitivity for early diagnosis if infants' mothers are receiving ART or the infants are receiving antiretroviral prophylaxis. The sensitivity of the test also may decline with increasing age. False-positive results do occur, and it usually is considered necessary to confirm positive results.( 21 )

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HIV DNA and RNA PCR

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HIV DNA PCR

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With the discovery of laboratory methods for amplifying DNA by PCR and the identification of HIV as the cause of AIDS, the HIV DNA PCR technique became simpler, less expensive, and more reliable than viral culture as a means for researchers to identify HIV-infected individuals and perform extensive epidemiologic studies.( 31 ) The availability of primers for HIV subtypes allowed researchers to use HIV DNA PCR to investigate and track HIV subtypes for vaccine development and epidemiological studies.( 32 )

HIV DNA PCR was first used for infant HIV diagnosis in a 1990 study that tested peripheral blood mononuclear cells (PBMC) from infants at various time points following birth.( 33 ) It was hoped that HIV DNA PCR would be as specific as viral culture in newborns but easier to perform, requiring less blood volume. Although DNA PCR did perform well, subsequent studies of newborns by Delamare et al ( 34 ) and Dunn et al ( 35 ) found that HIV DNA PCR detected <50% of infections within the first few days of life. Its sensitivity increased to 90% by 14 days of age.

The lack of sensitivity of HIV DNA PCR in diagnosing HIV infections at birth probably arises from the fact that most HIV infection of infants occurs during labor and delivery, and the virus does not reach detectable levels for several weeks after infection. Infants who are infected in utero may have only small amounts of replicating virus.

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HIV RNA PCR

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Attempting to find a method to diagnose infants earlier, researchers turned to HIV RNA PCR, which can detect HIV in plasma. Unlike HIV DNA PCR, which is a qualitative test (ie, it gives a yes/no HIV diagnosis), HIV RNA detection provides additional quantitative information about virological status, measuring the amount of circulating virus (otherwise known as "viral load" and expressed as copies/mL) in a patient. Thus, it can be used to diagnose patients, guide the initiation of ART, and monitor treatment response.

It was hoped that HIV RNA would be sensitive in viral detection while remaining highly specific for HIV, and that it would replace more complex and expensive viral culture techniques for infant diagnosis.( 36 ) Early studies in HIV-exposed infants using HIV RNA PCR assays found that the method matched or exceeded the sensitivity and specificity of HIV DNA PCR and viral culture methods.( 37 ) In a study by Lambert et al, sensitivity of HIV RNA PCR tests was 27% at birth, 92% at 6 weeks, and 91% at 20 weeks.( 38 ) Other investigators have reported similar results.( 39 , 40 , 41 )

Table 1. Sensitivities of HIV Culture, HIV DNA PCR, and HIV RNA PCR
Assay Birth 6 weeks 24 weeks
HIV culture 21% 90% 95%
HIV DNA PCR 10% 81% 81%
HIV RNA PCR 27% 92% 91%
Source: Lambert JS, Harris DR, Stiehm ER, et al. Performance characteristics of HIV-1 culture and HIV-1 DNA and RNA amplification assays for early diagnosis of perinatal HIV-1 infection. J Acquir Immune Defic Syndr 2003; 34 (5):512-9.
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HIV RNA testing equipment is becoming less expensive, and commercial assays for HIV RNA detection are now more widely available than DNA assays. However, this method does have several drawbacks, including a tendency to yield false-positive results for patients with low levels of viremia and the fact that not all primers and reagents are standardized.( 21 ) In addition, the increased use of ART and prophylaxis for PMTCT raises a potentially important issue regarding the sensitivity of HIV RNA PCR methodology for infant diagnosis. Antiretroviral drugs potentially could lower viral levels in peripheral blood mononuclear cells or plasma and decrease the sensitivity of these assays. However, studies indicate that the reduction in viral load occurring with ART or short-course antiretroviral prophylaxis does not interfere with PCR HIV detection methods.( 42 )

Summary

transparent gif bullet HIV DNA PCR is the most widely used assay in industrialized countries for early infant diagnosis.
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transparent gif bullet HIV RNA PCR has also been shown to be accurate and reliable, and it provides additional information about virological status.
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transparent gif bullet Both HIV DNA and HIV RNA technologies are becoming less expensive, more automated, and faster in producing results.
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Source: World Health Organization. Early detection of HIV infection in infants and children. Guidance note for development of round 6 GFTAM proposals and the Technical Review Panel to direct gap analysis and consideration of options for selection of technology for early diagnosis of HIV in infants in resource-limited settings.

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Virological Testing with DBS Samples

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Low-cost virological testing techniques remain elusive because of the high costs of performing RNA and DNA PCR, which is a direct result of restrictive intellectual property laws governing PCR technology. Given the high cost of PCR technologies, most countries wishing to scale up HIV infant diagnosis and viral load monitoring will be able to establish only a few central public laboratories with this capacity. The collection and transportation of blood samples to these central laboratories presents a major logistical hurdle. The use of DBS on filter paper instead of whole blood samples has been put forward as a means to mitigate the sample collection and transport challenges that programs might face when initiating large-scale virological testing.

With infants, the procedure for obtaining a DBS sample involves obtaining blood from a heel, toe, or finger stick and applying it directly onto filter paper, bypassing the need for needles, syringes, whole blood collection, and separation of blood into plasma. The amount of blood required is usually <75 µL. The use of DBS is attractive for resource-poor countries because blood samples on filter paper are easier to transport to centralized laboratory facilities for analysis than whole blood samples.( 43 ) Furthermore, mid-level health care workers in remote clinics who lack access to needles and syringes for collecting whole blood samples are able to perform heel sticks and similar procedures needed for DBS collection without a great deal of training. DBS samples are also safer to handle than whole blood samples, posing little biohazard risk. Samples can be stored up to a year at room temperature. DBS samples can be used for HIV RNA (viral load) tests, meaning that once a system for using DBS is in place, it could improve access to viral load measurements for adults and children.

DBS has been used in resource-rich settings for several decades in screening newborns for metabolic illnesses. The use of DBS for infant HIV diagnosis was initially evaluated in the United States because DBS samples already were being used to screen newborns for phenylketonuria. Cassol et al first demonstrated the reliability of DNA PCR testing on DBS samples in 1991.( 44 ) Subsequent studies of HIV DNA PCR on DBS have found that the method compares favorably with other HIV diagnostic tests such as viral culture and whole blood PCR.( 43 , 45 , 46 , 47 ) However, it is important to note that all of these diagnostic tests, whether or not they are performed on whole blood, have poor sensitivity when samples are taken from infants during the first week of life. The Women and Infants Transmission Study (WITS), which analyzed 272 samples from 144 infants during the first 4 months of life using DNA PCR, found that the sensitivity of the test within the first week of life was 19%. This increased to 96% by 1 month of age.( 48 , 49 ) Specimens obtained on the day of birth were least likely to have detectable HIV DNA on DBS. In this and other studies, the specificity of DNA PCR tests on DBS was greater than 99%.

Studies also have compared the performance of HIV DNA PCR tests using DBS to p24 antigen assays and viral culture using whole blood samples.( 45 , 50 , 51 , 52 ) As expected, the p24 antigen capture assays were relatively insensitive, whereas the HIV DNA PCR performed on DBS samples had an excellent correlation with viral culture.

Researchers have continued to simplify the technical procedure for performing PCR on DBS samples, allowing for more rapid analysis and standardization through routine laboratory methodology. Recent studies using these procedures have found that the method continues to perform well in high-volume clinical settings in resource-poor countries. For example, in South Africa, Sherman et al evaluated a commercially available DNA PCR technique on DBS samples collected on filter paper and found that the tests had a high specificity (99%) and sensitivity (100%), even with samples from infants as young as 6 weeks of age.( 43 )

In another seminal study, Bigger et al evaluated 15,810 filter paper cards using blood samples from infants in a large PMTCT study in southern Africa.( 53 ) Researchers collected blood samples from infants at age 6 and 12 weeks and conducted PCR testing in duplicate on each sample. They found that a positive PCR result after 1 month of age was 99% accurate in predicting a positive antibody test after 15 months.

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Virological Testing with DBS in Operational Settings

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Botswana

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Based on the encouraging results from research studies, several PMTCT programs have piloted the use of DBS for early infant diagnosis on a larger scale. One of the most prominent examples is Botswana's national PMTCT program, which has successfully piloted the use of DBS samples for testing infants at 6 weeks of age at 12 sites in Gaborone and Francistown and is now expanding the use of this method nationally.( 54 , 55 )

The use of DBS in the 6-month Botswana pilot was quite successful. The method was acceptable to both health care workers and mothers, and the collection of DBS samples was easily integrated into clinics' routine infant care.( 55 ) Health care workers received a 1-day classroom training on infant diagnosis followed by a hands-on training at each site. This one-on-one training was seen as being crucial in helping health care workers gain proficiency at obtaining samples.

DBS samples were sent by courier to the national HIV reference lab, where a technician performed PCR testing using the Roche Amplicor 1.5 test kit. Results were then transmitted back to the health care facilities by facsimile. More than 90% of babies born to HIV-infected mothers were tested in the pilot, with an average 9-day turnaround time from specimen collection to receipt of results at the clinic.( 55 ) Both nurses and midwives were able to collect DBS samples with little difficulty; nurses collected most samples (67%), and most infants (73%) only had to be stuck once to obtain a sample.

All positive results in were confirmed by a second test on same sample. There were no false positives detected in the pilot's antiretroviral clinics. The lab rejected only 1.7% of samples, mainly because of labeling errors; the quality of the samples was rarely a problem. The study authors suggest that programs explore creative ways to minimize labeling problems, for example by using names in addition to numbers to identify patients or by using cards attached to patient forms similar to those used for newborn screening in the United States.

Despite these excellent results, it is sobering to note that about a quarter of the HIV-infected children in this pilot were lost to follow-up, even with the availability of early HIV testing.

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Rwanda

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The International Center for AIDS Care and Treatment Programs (ICAP) at Columbia University's Mailman School of Public Health also has used DBS sampling in its HIV prevention, care, and treatment programs in resource-poor settings. At the XIV International AIDS conference in Toronto in August 2006, Dr. Luis Felipe Gonzales described ICAP's experience in successfully piloting PCR testing for early infant diagnosis, providing an outline of the steps needed to set up an infant diagnosis network.( 56 )

As in Botswana, the Rwanda program used the Roche Amplicor 1.5 test kit to perform DNA extraction from DBS samples. Analyzing the results from 345 samples, the investigators found that the tests had good sensitivity (98.02%) and specificity (100%), and compared well with whole blood samples. They found only 1 false-negative sample.

An interesting finding of the Rwanda program was that the main challenge in expanding early infant diagnosis was not conducting PCR tests at the laboratory level but rather ensuring that children were properly followed up at the clinic level. The investigators found that each clinic needed a focal person, usually a nurse, who would be in charge of guiding families through the HIV testing procedure and subsequent care and treatment. This involved tasks such as taking the infant DBS sample, packaging and sending the DBS to the central lab, making sure that the clinic received the results, putting the results in the child's file, and making clinical decisions based on results and the clinic's protocols.( 56 )

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Challenges to Using DBS Samples for Early Infant Diagnosis

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Investigators using DBS for PCR testing in resource-poor settings have encountered several challenges, including difficulty storing filter paper samples correctly, errors in labeling of blood samples, problems transporting filter paper samples, testing delays when the DBS samples are transferred to a central laboratory, lack of quality control, and cross-contamination of samples. Many of these problems are inherent to all PCR techniques. However, by far the largest obstacle to using DBS samples with PCR for early infant diagnosis is the cost of the PCR assays, which averaged US$10 to $15 in 2005.( 57 ) In contrast, rapid antibody tests are currently available at US$2 to $4 per test.

Also, as the experience in Rwanda shows, the presence of health care workers at the clinic level who are trained to take samples and follow up children is as important to the implementation of early infant HIV diagnosis as the development of central laboratories. It should be noted that, in many countries, only laboratory technicians are authorized to draw blood, and such restrictions continue to limit the ability of programs to scale up HIV diagnostic and monitoring services for children and adults.

In sum, although studies of DBS HIV PCR are encouraging, without simplification of the procedure, a reduction in the cost of PCR reagents and equipment, and a continuation of efforts to increase the number of trained health care workers in facilities, the potential of this method to bring about a significant increase in access to early HIV diagnosis for infants is limited.

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General Recommendations for Scaling Up Virological Testing for Early Infant HIV Diagnosis (58)

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transparent gif bullet Because perinatally infected infants face a high risk of mortality, and because pediatric ART is increasingly available, national PMTCT programs in resource-poor countries should build the capacity to provide virological HIV testing for infants.
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transparent gif bullet Antibody assays suitable for diagnosing HIV infection in adults cannot be used to diagnose infection in infants under 18 months of age reliably. However, antibody tests can be used to screen children in need of further virological testing. Draft WHO guidelines recommend screening children at 9 months, at which time more than half of the potential maternal antibodies will have dissipated.( 21 )
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transparent gif bullet Virological testing for HIV-exposed infants should be performed at 6 weeks of age. Testing at that time gives a good sensitivity (>98%) with the various PCR methods and will identify most of the infants infected in utero and during labor and delivery. These are the infants at greatest risk of rapid disease progression and in the most urgent need of care and treatment.
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transparent gif bullet Whenever possible, a positive PCR result should be confirmed using the same specimen or, if feasible, a second specimen obtained 1-2 weeks after the first one.
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transparent gif bullet Decisions about the timing of testing and repeat testing should be based in part on the breast-feeding practices that are prevalent in a particular region. The risk of acquiring HIV infection from the mother continues through the entire breast-feeding period, and breast-fed children who have a negative result on their initial test will need to be retested 6 weeks after the cessation of breast-feeding.
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transparent gif bullet The clinical presentation of the infant should be considered in the interpretation of virologic and antibody test results.
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transparent gif bullet HIV testing protocols should have provisions for children who do not come into programs through PMTCT activities but who present at health care facilities with signs and symptoms of HIV infection. Currently, this is how the bulk of pediatric HIV cases are found. Rapid antibody tests can be used to screen these children for further virological testing.
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transparent gif bullet National PMTCT program planners should aim to develop laboratory capacity for performing virological tests using HIV DNA or RNA PCR at the central or tertiary level. They should try to achieve national coverage through the development of laboratory networks and referral systems.
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transparent gif bullet The sensitivity of DNA PCR can vary according to the type of assay used and the laboratory that performs the tests; national programs should be designed to ensure that the assays in use are sensitive to the viral subtypes prevalent in a particular region.
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transparent gif bullet National programs should incorporate PCR technology that supports the use of DBS samples. To improve cost effectiveness, program planners should consider selecting PCR platforms that can be used to measure other infections such as hepatitis B, hepatitis C, and Mycobacterium tuberculosis .
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Because of the cost and complexity of the technologies, laboratory managers need careful long-term planning regarding the development of standard operating procedures; procurement plans; delivery, storage, and stock management systems; external and internal quality assessment schemes; training; and facility management structure. Programs planners must carefully estimate the number of infants that will require testing in order to choose the most efficient testing equipment and to ensure that it will not be idle for long periods of time.

Factors to consider when choosing PCR technology:

transparent gif bullet New purchase of required equipment
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transparent gif bullet Commercial availability and cost of equipment and reagents
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transparent gif bullet Projected numbers of tests from specific geographical areas
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transparent gif bullet Projected number of samples to be processed
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transparent gif bullet Specimen storage and transport
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transparent gif bullet Ongoing laboratory quality assurance
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transparent gif bullet Availability of maintenance and service for equipment and supplies
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transparent gif bullet Sample collection and processing (including DBS specimen to machine)
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transparent gif bullet Other equipment required (eg, automated specimen preparation, DBS punchers, centrifuge)
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transparent gif bullet Viral types and subtypes
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transparent gif bullet Training and availability of laboratory staff
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transparent gif bullet Use of the equipment for other purposes (eg, diagnosis of other conditions or monitoring of ART)
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Source: World Health Organization. Early detection of HIV infection in infants and children. Guidance note for development of round 6 GFTAM proposals and the Technical Review Panel to direct gap analysis and consideration of options for selection of technology for early diagnosis of HIV in infants in resource-limited settings.

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Summary

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Despite the potential of PCR methodologies, their costs remain prohibitive for resource-poor countries. Further reduction in the costs of PCR technology, corporate programs for donation of supplies and equipment, and the transfer of intellectual property rights to generic manufacturers (as has occurred with patented drugs) would greatly facilitate implementation of low-cost early diagnosis of HIV infection in infants.

Low-cost HIV infant diagnosis and the availability of pediatric ART are closely linked. Early HIV diagnosis has a greater impact on public health outcomes and expenditures when it is coupled with improved access to ART. The advocacy that has resulted in widespread price reductions of patented drugs for adults and children also could be effective in reducing the costs of HIV diagnosis through more open interpretation of intellectual property laws in dealing with life-threatening diseases such as HIV. Innovative new approaches are required if the cost of current diagnostic testing cannot be reduced. As Aledort et al state, "There is an urgent need to develop and deploy a new, easy-to-use HIV test, which could transform the management of pediatric HIV/AIDS in developing countries and avert millions of infant deaths."( 11 )

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References

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  1. World Health Organization. Antiretroviral Drugs for Treating Pregnant Women and Preventing HIV Infection in Infants: Guidelines on Care, Treatment and Support for Women Living With HIV/AIDS and their Children in Resource-Constrained Settings. Geneva: World Health Organization . July 2004.
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