Pediatric HIV Infection Workup

Prompt diagnosis of human immunodeficiency virus (HIV) infection is critical. As such, the Centers for Disease Control and Prevention (CDC) recommends routine prenatal HIV testing as the standard of care for all pregnant women in the United States, with repeat screening in the third trimester recommended in certain jurisdictions with elevated rates of HIV infection among pregnant women.^[28]

Diagnosis of HIV infection in infants is aided by HIV culture or DNA/RNA polymerase chain reaction (PCR); positive results are confirmed by repeating the test. In suspected cases, HIV testing should occur in the newborn period (ie, before the infant is 48 h old), at age 1-2 months, and again at age 3-6 months. Testing at age 14 days may allow for earlier detection of HIV in infants who had negative test results within the first 48 hours of life. By approximately age 1 month, PCR testing has a 96% sensitivity and 99% specificity to identify HIV.

The 2010 Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children^[29] recommendations for diagnosing infants are as follows:

• Because of the persistence of the maternal HIV antibody, infants younger than 18 months require virologic assays that directly detect HIV in order to diagnose HIV infection
• Preferred virologic assays include HIV bDNA PCR and HIV RNA assays
• Virologic diagnostic testing is recommended at birth in infants at high risk of HIV infection (eg, infants born to HIV-infected mothers who did not receive prenatal care or prenatal antiretroviral therapy or who had HIV viral loads ≥1,000 copies/mL close to time of delivery)
• Further virologic testing in infants with known perinatal HIV exposure is recommended at 14-21 days, at 1–2 months, and at 3–6 months.
• An antibody test to document seroreversion to HIV antibody–negative status in uninfected infants is recommended at age 12–18 months
• In children 18 months and older, HIV antibody assays can be used for diagnosis

The Panel does not recommend use of the currently approved HIV p24 antigen assay for infant diagnosis in the United States because the sensitivity and specificity of the assay in the first months of life is less than that of other HIV virologic tests.

Within the first 48 hours, 14 days, and 4 weeks of life, 38%, 93%, and 96% of infected children, respectively, have positive HIV DNA PCR results. Any positive HIV DNA PCR finding should be confirmed with follow-up HIV DNA PCR before infection is diagnosed.

HIV infection can be ruled out if one of the following is true:

• DNA HIV PCR results are consistently negative in an infant older than 4 months in the absence of breastfeeding.
• Two DNA HIV PCR results obtained at least a month apart are negative in an infant older than 6 months.

Monitor CD4⁺ levels or percentages in infants or patients newly diagnosed with HIV at 3- to 4-month intervals to assess patients' immune status. In children younger than 5 years, the 2010 Panel recommends using CD4 percentages over absolute CD4 counts for monitoring disease progression because of inherent age-related changes in absolute CD4 counts.

Monitor for opportunistic infections. Perform a CBC count with differential and a urinalysis every 1-3 months in infants. Older children can be screened every 3-6 months (CBC count) or yearly (urinalysis). Culture urine samples monthly for the presence of cytomegalovirus (CMV) until age 2 months and then at 2-month intervals until age 12 months.

Assess HIV RNA levels twice at baseline and then every 3-4 months. (Consistently use the same HIV RNA assay method to monitor a particular patient.) More frequent testing of HIV RNA levels and CD4 counts may be necessary for children who have virologic or clinical deterioration or when initiating or changing antiretroviral therapy or in children under 12 months of age.

If the mother is HIV positive, use serologic tests to screen the infant for hepatitis B, hepatitis C, syphilis, and toxoplasmosis.

Decreased levels of albumin, serum immunoglobulin G, and CD8⁺ T cells are linked with fatality in children.

Monitor laboratory studies in accordance with drug therapy protocols and clinical status (eg, lipid profile in a patient with lipodystrophy).

Perform chest radiography, CT scanning, MRI, echocardiography, and electrocardiography for baseline determinations and subsequently as clinically indicated. For example, brain imaging is indicated in patients with suspected HIV encephalopathy. Imaging may demonstrate degeneration of the white matter, atrophy, and/or calcifications of the basal ganglia with progressive HIV encephalopathy.

HIV detection is the first step in the laboratory workup. In September 2006, the CDC released its Revised Recommendations for HIV Testing of Adults Adolescents, and Pregnant Women in Health-Care Settings.^[28] These new recommendations, which replaced the CDC’s 1993 Recommendations for HIV Testing Services for Inpatients and Outpatients in Acute Care Hospital Settings, advise routine HIV screening for adults, adolescents, and pregnant women in healthcare settings in the United States. They also recommend reducing barriers to HIV testing.

A 2011 revision to the American Academy of Pediatrics (AAP) policy statement recommends that routine screening be offered to all adolescents at least once by 16 to 18 years of age in communities with high HIV prevalence. In areas of lower prevalence, routine testing is encouraged for all sexually active adolescents and those with other risk factors for HIV.^[30]

Detection of antibody to HIV is the usual first step in diagnosing HIV infection. In adults and older children, enzyme-linked immunosorbent assay (ELISA) and Western blotting are used to initially detect HIV-specific antibodies. However, because maternal antibodies are present in neonatal blood, these tests are not used for diagnosis in patients younger than 2 years. A nucleic acid PCR assay is the standard detection method in infants and young children.

HIV DNA PCR is used to detect HIV-1 provirus in mononuclear cells by using oligonucleotides directed at highly conserved regions of the viral genome. This test can be performed within 24 hours of infection and has a sensitivity and a specificity of 95% and 97%, respectively. Although it is more sensitive than viral culturing, the diagnostic performances of the 2 methods are equivalent.

Viral cultures are obtained by co-cultivating potentially infected and uninfected mononuclear cells together to promote viral replication. Every few days, the culture is assayed for HIV p24 antigen. Positive results on 2 sequential p24 antigen detection assays indicate infection. This technique requires a mean of 7-14 days to perform, but it may require as long as 28 days.

A positive virologic result should be confirmed with repeat virologic testing with a second specimen as soon as possible after the first result is available.

ELISA for HIV antibody, followed by a confirmatory Western blot (which has increased specificity), should be used to diagnose HIV infection in older children and adults.

Rapid HIV tests, which provide results in minutes, simplify and expand the availability of HIV testing. Their sensitivity is as high as 100%, but they must be followed with confirmatory Western blotting or immunofluorescence antibody testing, as with conventional HIV antibody tests.

The US Food and Drug Administration (FDA) has approved 4 rapid HIV screening tests that are available commercially in the United States, as listed below. Before the FDA approved these tests, the most commonly used rapid HIV test was the Single-Use Diagnostic System (Murex SUDS; Abbott Diagnostics, Abbott Park, IL), which is no longer available.

• OraQuick (and its newer version OraQuick Advance) Rapid HIV-1/2 Antibody Test (OraSure Technologies, Inc, Bethlehem, PA)
• Reveal G3 Rapid HIV-1 Antibody Test (MedMira Laboratories, Inc, Halifax, Nova Scotia, Canada)
• Uni-Gold Recombigen HIV Test (Trinity Biotech, Bray, Ireland)
• Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories, Redmond, WA)

HIV RNA assays are used to detect extracellular viral RNA in the plasma and are as sensitive as HIV DNA PCR for early diagnosis of HIV infection in exposed infants. However, nucleic acid amplification tests are not the preferred methods of diagnosis in infants perinatally exposed because they may falsely indicate low viral loads in individuals who are HIV negative. Also, whether antenatal treatment of the mother with combination antiretrovirals and/or antiretroviral prophylaxis in the infant affects the sensitivity of RNA assays is unknown.

The FDA has approved the APTIMA HIV-1 RNA Qualitative Assay (Gen-Probe Incorporated, San Diego, CA). This assay enables people to learn with certainty whether they are infected within a few days rather than up to 6 months, as with an HIV antibody test. Unlike some approved antibody tests, the APTIMA assay is intended to detect only HIV-1 and not HIV-2.

The test could become a potential alternative to traditional Western blotting now used to confirm HIV-1 infection when screening results for HIV antibodies are positive. The APTIMA assay is approved for the diagnosis of primary HIV-1 infection for confirming HIV-1 infection when antibody results are positive.

The viral load can be quantified by using several HIV assays. The number of virions in the peripheral blood is an important indicator of disease activity and of the effectiveness of antiretroviral therapy (ART). A 5- or 3-fold change in the viral load is needed to reliably indicate a clinically significant change in children younger than 2 years or older than 2 years, respectively.

Certain viral-load tests are not sensitive to non-B subtypes of HIV-1. Therefore, viral loads can seem to be considerably reduced if these tests are used to process samples of non-B subtypes.

Reverse-transcription PCR (RT-PCR) and nucleic acid sequence—based amplification (NASBA) of plasma RNA reveal a viral load 2 times that obtained with the branched-chain DNA (bDNA) method. The former methods are sensitive to only HIV-1 subtype B viruses, whereas the bDNA method is sensitive to other HIV-1 subtypes. Switching test methods during treatment is not advised because their molecular technologies differ.

Initial infection is associated with high viral loads, especially in the neonate. In adults and adolescents with nonvertically acquired infections, the viral load rapidly decreases 6-12 months after the primary viremia. Neonates have high viral loads that persist throughout infancy; therefore, these values are hard to interpret in the first year of life. In children with vertically acquired infections, the load slowly declines after 1 year of age. The predictive value of specific HIV RNA concentrations for disease progression and death for an individual child in the first year of life is moderate.

Viral resistance to ART may be present. Both primary and secondary mutations can develop. Primary mutations alter the effectiveness of ART. Secondary mutations improve viral survival.

Both genotypic and phenotypic assays can be performed. Genotypic assays are fast and available, but they reveal only known mutations, and they cannot be used to predict complex interactions when several antiretroviral drugs (ARDs) are used together.

Automated recombinant phenotypic assays are commercially available, but the results require additional time to be ready, and the tests are expensive. However, these assays can be used to detect complex interactions between ARDs and quasispecies, to perform in vitro drug trials, and to measure ART inhibitory concentrations.

Resistance testing is recommended before the initiation of ART in all treatment-naïve children, with the results used to refine selection of drugs for initial combination therapy. Resistance testing is also recommended before changing therapy for treatment failure.^[29]

Genotype and phenotype assays may not be useful for detecting minor quasispecies, and treatment failure occurs despite the use of these techniques.

Development of resistance to 1 or more of the drugs in a combination regimen is often the cause for viral rebound. Numerous clinical trials have demonstrated the clinical use of ARD-resistance testing in selecting an alternate effective combination and in improving clinical outcomes.

The CD4⁺ lymphocyte count is a surrogate marker for disease progression and should be monitored closely. The CD4⁺ count should be obtained before therapy. A rapid decrease in the count, especially in infants younger than 1 year, is a poor prognostic sign and should prompt the start or alteration of therapy.

Consumptive thrombocytopenia is a common finding in children with HIV infection and may be observed in 10% of patients at initial diagnosis.

Anemia occurs in as many as 20% of patients at diagnosis and occurs in as many as 80% of patients at some time. Anemia can have many etiologies in HIV infected individuals and requires a workup as described in Medical Care.

A high mean corpuscular volume (MCV) is most commonly caused by zidovudine and can be used to verify compliance. Other medications also cause a high MCV, as well as vitamin B-12 and folate deficiencies.

Anemia continues to predict decreased survival even with highly active ART (HAART).

Pancytopenia results from folate deficiency, use of pharmaceutical agents, and infections with viruses such as parvovirus B19.

Neutropenia is observed in 10% of patients with early asymptomatic HIV infections and in 50% of patients with AIDS.

Blood smears may reveal large ovalocytes and hypersegmented polymorphonucleocytes in cases of folate deficiency.

Serum electrolytes should be monitored on a regular basis because medications or HIV infection may induce nephrotoxicity.

Liver function can be impaired as a result of medication, HIV, co-infection with hepatitis viruses, or opportunistic infections, so transaminase levels should be monitored. Pancreatitis can be the result of medication, HIV or opportunistic infections, so amylase and lipase levels should be monitored in patients with abdominal symptoms. Parotiditis (parotitis) is not uncommon, and amylase levels should be followed up if parotiditis is suspected or if the patient has a history of the condition.

Quantitative immunoglobulin levels should be followed up periodically. Hyperimmunoglobulinemia is associated with disease progression. Hypoimmunoglobulinemia is observed in end-stage disease and is associated with a poor prognosis.

Patients with HIV nephropathy demonstrate increased size and echogenicity of the kidneys on renal ultrasonography, with a loss of cortical medullary differentiation. Renal cysts are observed with an increased incidence.

On renal CT scanning, stasis of urine in the pyramids is observed in patients with HIV nephropathy. This finding, combined with characteristic renal ultrasound findings, is specific for HIV nephropathy.

Renal scintigraphy with technetium-99m mercaptoacetyltriglycine (^99m Tc MAG3) demonstrates delayed elimination of the tracer and increased residual activity consistent with tubular dysfunction in HIV nephropathy. These abnormalities correlated with creatinine clearance.

On renal gallium scanning, increased signal indicates inflammation in patients with HIV nephropathy and is correlated with proteinuria.

A biopsy sample should be taken from enlarged lymph nodes of undetermined cause, especially if they are single, hard, nonmotile, or unaccompanied by generalized lymphadenopathy.

Biopsy may also be considered to clearly determine the identity of an apparently infectious or malignant cutaneous lesion. Maintain a high index of suspicion for a wide array of infections and malignancies, and request the appropriate staining and tissue preparation.

Lymphoid interstitial pneumonitis (LIP) is the second most common AIDS-defining illness in children. LIP most commonly occurs in children with a relatively high CD4⁺ count. Chest radiography demonstrates a reticulonodular pattern with or without hilar adenopathy that persists for more than 2 months despite treatment.

Patients are usually asymptomatic at first, but cough and shortness of breath develop as lymphoid interstitial pneumonitis progresses. Hypoxia typically responds to a 2-week course of steroids, but oxygen dependence develops if an underlying chronic lung disease exists. Lymphoid interstitial pneumonitis increases the risk of bacterial pneumonia, especially with Haemophilus influenzae and pneumococcus.

Recurrent pneumonia destroys lung tissue and leads to chronic lung disease. Chest radiographs demonstrate chronic changes, including areas of chronic atelectasis. This condition requires management by a pulmonologist. Chronic respiratory therapy may be required, including home oxygen therapy.

Clinical categories are based on the 2010 CDC guidelines^[29] for antiretroviral treatment of pediatric AIDS (review and modification of the 1994 CDC HIV pediatric classification system for clinical categories in children younger than 13 y). This system uses a clinical-category letter and an immunologic number to note each stage of disease progression. The clinical categories are based on clinic manifestations. The immunologic category is based on the age-dependent CD4⁺ count (see Table 1, below).

Clinical categories include the following:

• N – Not symptomatic
• A – Mildly symptomatic
• B – Moderately symptomatic
• C – Severely symptomatic.

Category N includes children who have no signs or symptoms considered to be the result of HIV infection or who have only one of the conditions listed in category A.

Category A includes children with 2 or more of the following conditions but none of the conditions listed in categories B and C:

Lymphadenopathy (≥0.5 cm at more than 2 sites; bilateral = one site)

• Hepatomegaly
• Splenomegaly
• Dermatitis
• Parotitis
• Recurrent or persistent upper respiratory infection, sinusitis, or otitis media

Category B includes children who have symptomatic conditions, other than those listed for category A or category C, that are attributed to HIV infection. Examples of conditions in clinical category B include, but are not limited to, the following:

• Anemia (< 8 g/dL), neutropenia (< 1,000 cells/µL), or thrombocytopenia (< 100,000 cells/µL) persisting 30 days or longer
• Bacterial meningitis, pneumonia, or sepsis (single episode)
• Candidiasis, oropharyngeal (ie, thrush) persisting for greater than 2 months in children older than 6 months
• Cardiomyopathy
• Cytomegalovirus infection with onset before age 1 month
• Diarrhea, recurrent or chronic
• Hepatitis
• Herpes simplex virus (HSV) stomatitis, recurrent (ie, >2 episodes within 1 y)
• HSV bronchitis, pneumonitis, or esophagitis with onset before age 1 month
• Herpes zoster (ie, shingles) involving at least 2 distinct episodes or more than one dermatome
• Leiomyosarcoma
• Lymphoid interstitial pneumonia (LIP) or pulmonary lymphoid hyperplasia complex
• Nephropathy
• Nocardiosis
• Fever lasting greater than 1 month
• Toxoplasmosis with onset before age 1 month
• Varicella, disseminated (ie, complicated chickenpox)

Category C includes children who have any condition listed in the 1987 surveillance case definition for AIDS, with the exception of LIP (which is a category B condition). These conditions are as follows:

• Serious bacterial infections, multiple or recurrent (ie, any combination of at least 2 culture-confirmed infections within a 2-y period), of the following types: septicemia, pneumonia, meningitis, bone or joint infection, or abscess of an internal organ or body cavity (excluding otitis media, superficial skin or mucosal abscesses, and indwelling catheter-related infections)
• Candidiasis, esophageal or pulmonary (bronchi, trachea, lungs)
• Coccidioidomycosis, disseminated (at site other than or in addition to lungs or cervical or hilar lymph nodes)
• Cryptococcosis, extrapulmonary
• Cryptosporidiosis or isosporiasis with diarrhea persisting greater than 1 month
• Cytomegalovirus disease with onset of symptoms at older than 1 month (at a site other than liver, spleen, or lymph nodes)
• Encephalopathy (at least one of the following progressive findings present for at least 2 mo in the absence of a concurrent illness other than HIV infection that could explain the findings): Failure to attain or loss of developmental milestones or loss of intellectual ability, verified by standard developmental scale or neuropsychological tests; impaired brain growth or acquired microcephaly demonstrated by head circumference measurements or brain atrophy demonstrated by CT scanning or MRI (serial imaging required for children < 2 y); acquired symmetric motor deficit manifested by 2 or more of the following: paresis, pathologic reflexes, ataxia, or gait disturbance
• Herpes simplex virus infection causing a mucocutaneous ulcer that persists for greater than 1 month; or bronchitis, pneumonitis, or esophagitis for any duration affecting a child older than 1 month
• Histoplasmosis, disseminated (at a site other than or in addition to lungs or cervical or hilar lymph nodes)
• Kaposi sarcoma
• Lymphoma, primary, in brain
• Lymphoma, small, noncleaved cell (Burkitt), or immunoblastic or large cell lymphoma of B-cell or unknown immunologic phenotype
• Mycobacterium tuberculosis, disseminated or extrapulmonary
• Mycobacterium, other species or unidentified species, disseminated (at a site other than or in addition to lungs, skin, or cervical or hilar lymph nodes)
• M avium complex or M kansasii, disseminated (at site other than or in addition to lungs, skin, or cervical or hilar lymph nodes)
• Pneumocystis jiroveci pneumonia
• Progressive multifocal leukoencephalopathy
• Salmonella (nontyphoid) septicemia, recurrent
• Toxoplasmosis of the brain with onset at older than 1 month
• Wasting syndrome in the absence of a concurrent illness other than HIV infection that could explain the following findings: Persistent weight loss of >10% of baseline OR Downward crossing of at least 2 of the following percentile lines on the weight-for-age chart (eg, 95th, 75th, 50th, 25th, 5th) in a child aged 1 year or older OR Less than the 5th percentile on weight-for-height chart on 2 consecutive measurements, 30 days or greater apart PLUS 1) chronic diarrhea (ie, ≥2 loose stools/d for >30 d) OR 2) documented fever (for ≥30 d, intermittent or constant)

Once an advanced class is assigned, the disease cannot be reassigned to a lesser class, even if the clinical or immunologic manifestations resolve. Category C and clinical manifestations of advanced disease are synonymous with AIDS.

Table 1. CDC Immunologic Categories for HIV-Infection in Children Based on Absolute CD4⁺ Counts (Open Table in a new window)