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Overview of 2010 WHO Guidelines for Early Infant HIV Diagnosis

The diagnosis of HIV infection in infants is of paramount importance since HIV disease typically progresses much more rapidly in infants than it does in adults[1,2,3]. Among infants who become infected with HIV and do not receive antiretroviral treatment, an estimated one-third will die by 1 year of age and approximately one-half will die by 2 years of age[4]. Accordingly, the 2010 WHO Antiretroviral Therapy for HIV Infection in Infants and Children guidelines emphasize the importance of early diagnosis in HIV-exposed infants, and recommend immediate initiation of antiretroviral therapy for all infants and children 2 years of age or younger who are diagnosed with HIV infection[4]. The following discussion will address approaches to HIV testing in infants and the specific diagnostic tests recommended in this setting.

Use of Serologic (HIV Antibody) Tests in the Diagnosis of Infants and Children

The use of HIV antibody testing (generally referred to as serologic testing in the WHO guidelines) to identify the presence of antibodies directed against HIV is an effective way to diagnose HIV infection in adults and children greater than 18 months of age. The approach to diagnosing HIV infection in infants and children younger than 18 months of age is different than with older children and adults, primarily because maternal anti-HIV antibodies diffuse across the placenta during gestation and can persist in the infant's bloodstream until at least 15 months of age. Thus, a positive HIV antibody test in an infant does not necessarily indicate that the infant is infected with HIV.

Use of Virologic Assays (DNA or RNA PCR) in the Diagnosis of Infants and Children

Virologic testing—measurement of HIV RNA or DNA using Polymerase Chain Reaction (PCR)—is the preferred method of infant HIV diagnosis, and should ideally be performed at 4 to 6 weeks of age in all HIV-exposed infants. The difficulty in differentiating HIV-exposed from HIV-infected infants due to the presence of maternal anti-HIV antibodies does not apply to virologic testing, since viral nucleic acid is directly detected. When performed at 4 to 6 weeks of age, virological testing will identify 95% of infants infected in utero or intrapartum[5,6]. Because most perinatal infections occur during the peripartum period, specimens collected from an HIV-infected infant immediately after birth may not yet be positive, since detectable HIV levels typically appear 7 to 10 days after acquisition of HIV. Although virologic testing on specimens collected within 48 hours of delivery will correctly diagnose most infants infected with HIV in utero, infants infected in the peripartum period generally do not have detectable viremia until 1 to 2 weeks after delivery[5]. Despite the relatively high sensitivity and specificity of properly performed HIV virologic assays, false positive and false negative test results can and do occur. Because the consequences of incorrect diagnoses are so great (lifelong antiretroviral therapy with a false-positive test or the hazard of death due to a missed HIV diagnosis), confirmatory testing is necessary and should be performed as soon as possible. Importantly, an initial positive result should prompt immediate initiation of antiretroviral therapy and treatment should not be deferred while awaiting the results of confirmatory testing. The WHO has developed an infant HIV testing algorithm that is now widely used in resource-limited settings (Figure 1)[4].

Testing of Infants whose HIV Exposure History is Not Known

On first contact with the health system, all infants should have their HIV exposure status established, preferably through maternal questioning or review of records. If maternal testing has not been done, or is unavailable, serologic testing should be performed on the mother after obtaining informed consent. If the mother is not available, or does not consent to maternal HIV testing, then serological testing of the infant may be appropriate, provided that parental or guardian consent to test the infant is obtained. In areas where the HIV epidemic is generalized (greater than 1% national seroprevalence), guidelines strongly recommend that all health centers establish HIV exposure status on all infants before 6 weeks of age by providing maternal and/or infant serological testing [4]. Virologic testing at 4 to 6 weeks of age (or at the earliest opportunity thereafter) should be performed on any infant with known exposure to HIV. If an infant of unknown HIV status should present to a health facility with signs, symptoms, or medical conditions that suggest possible HIV infection, serological testing should be performed first. Those with detectable HIV antibodies should then undergo virologic testing to confirm HIV infection [4].

Dried-Blood Spot (DBS) Specimen Collection

HIV DNA PCR assays can be performed on whole blood or dried bloodspots and HIV RNA PCR assays can be performed on plasma or dried bloodspot specimens. Dried bloodspot-based testing is generally preferred in resource-limited settings due to advantages in collection, storing, and processing. Dried bloodspot specimens do not require venipuncture, but rather can be obtained by finger or heel-stick method using simple blotting onto standardized filter paper forms (Figure 2). The dried bloodspot-based testing method poses a smaller biohazard risk than liquid samples, they are stable at room temperature for prolonged periods, and are easier to transport than liquid samples (Figure 3).

HIV Testing of Breastfeeding Infants

In resource-limited settings, many infants will continue breastfeeding throughout infancy and even beyond 15 months of age. The 2010 WHO guidelines recommend exclusive breastfeeding for 6 months and continuation of breastfeeding through 12 months[7]; breastfeeding should not be stopped in order to perform any kind of diagnostic HIV test [8]. A positive virologic HIV test result in a breastfeeding infant should be taken to indicate HIV infection, and the infant should start on antiretroviral therapy as soon as possible while repeating virologic HIV testing to confirm the initial positive result. In contrast, interpreting a negative test is different, due to the ongoing risk of HIV transmission via breastfeeding that continues throughout the breastfeeding period, even if the mother and/or infant are receiving appropriate antiretroviral prophylaxis. Therefore, a negative virologic test can only be interpreted as reliably negative if the infant blood sample was obtained more than 6 weeks after the cessation of breastfeeding, regardless of the age of the infant or child. According to the WHO infant HIV testing algorithm (Figure 1), infants who have a negative virologic test at 4 to 6 weeks of age, but who continue to breastfeed, should undergo serologic testing at 9 months of age[4]. Positive serologic tests should be followed by confirmatory virologic testing to differentiate HIV-exposed from HIV-infected infants. Negative serologic tests should be repeated at least 6 weeks after complete cessation of breastfeeding and/or at 18 months of age.

Rationale for Early Antiretroviral Therapy for HIV-infected Infants and Young Children

Observational studies have demonstrated unacceptably high mortality in cohorts of HIV-infected infants and children; in the absence of antiretroviral treatment, mortality rates are estimated to be 35 to 40% prior to 1 year of age [9,10], and one study demonstrated a 50% mortality before 2 years of age (Figure 4)[10]. A subsequent randomized controlled trial confirmed that immediate initiation of antiretroviral therapy in HIV-infected infants, regardless of clinical and immunological status, resulted in significantly lower mortality when compared with deferral of antiretroviral until clinical or immunological criteria for initiation were met (Figure 5)[1]. The WHO therefore now recommends antiretroviral therapy for all HIV-infected infants and children younger than 2 years of age, regardless of CD4 count, CD4 percentage, or clinical status[4].


Early infant diagnosis of HIV infection is essential to identify infected children and to prioritize them for early anti-retroviral therapy. The diagnosis of HIV in infants using serologic methods is complicated by the presence of maternal anti-HIV IgG antibodies, which cross the placenta and may persist through the first 15 months of life. For early infant HIV diagnosis, direct detection of HIV using virologic assays is preferred over serological assays. The ideal time to perform virologic assay is at 4 to 6 weeks of age because more than 95% of infants infected in utero or in the peripartum period will have positive results by this time. For breastfeeding infants, testing is complicated by the ongoing risk of HIV transmission throughout the breastfeeding period. Ruling out HIV infection requires a negative serologic test at least 6 weeks after the cessation of breastfeeding. In resource-limited settings, dried bloodspots on filter paper are the preferred method of specimen collection for virologic testing due to reliability and convenience of transporting specimens across large distances. Systems should be put in place to ensure rapid delivery of test results to the mother/infant pair. Any infant with an initial positive virologic test result should receive antiretroviral therapy without delay, while repeat virologic testing is performed to confirm the diagnosis.

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    Figure 1. WHO Early Infant Diagnosis Algorithm

    This flowchart summarizes approaches to diagnosing HIV infection in infants, including diagnostic options in settings where virologic assays are not available.

    Source: WHO. Antiretroviral therapy of HIV infection in infants and children: towards universal access. Recommendations for a public health approach – 2010 revision. World Health Organization. Geneva, Switzerland.

    Figure 1
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    Figure 2. Collection of Dried Bloodspot (DBS) Samples on Filter Paper

    A simple heel or toe prick can be used to obtain whole blood that is applied directly to filter paper and allowed to dry.

    Source: Christian Ramers, MD MPH

    Figure 2
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    Figure 3. Dried Blood Spot (DBS) Samples in a Rural Health Facility Awaiting Delivery to a Central Laboratory

    Dried blood spot (DBS) samples do not require refrigeration and pose minimal biohazard risk as compared with conventional whole blood or plasma samples.

    Source: Christian Ramers, MD MPH

    Figure 3
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    Figure 4.Increased Early Mortality in HIV-infected Infants in South Africa

    This observational study involving HIV-exposed infants in South Africa demonstrated a very high mortality rate in HIV-infected infants who did not receive antiretroviral therapy. By the age of 1 year, 35.2% of HIV-infected and 4.9% of HIV-uninfected children died. By the age of 2 years, 52.5% of HIV-infected and 7.6% of HIV-uninfected children died. Source: Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F; Ghent International AIDS Society (IAS) Working Group on HIV Infection in Women and Children. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet. 2004;364:1236-43.

    This figure reproduced with permission from Elsevier. Copyright © 2004 Elsevier. All rights reserved.

    Figure 4
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    Figure 5. Early Antiretroviral Therapy and Mortality among HIV-Infected Infants: the CHER Trial

    The CHER trial randomized 377 South African HIV-infected infants 6-12 weeks of age with CD4% of 25% or greater to either initiate antiretroviral therapy immediately ("early treatment") or defer therapy until immunologic and/or clinical criteria for ART initiation were met ("deferred treatment"). After a median of 40 weeks of follow-up, 16% of the infants in the deferred treatment arm had died, as compared with just 4% of the infants in the early treatment arm.

    Source: Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med. 2008;359:2233-44.

    This figure reproduced with permission from the Massachusetts Medical Society. Copyright © 2008 Massachusetts Medical Society. All rights reserved.

    Figure 5