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Perinatal HIV infection: Diagnostic issues
86 C L I N I C A L I M M U N O L O G Y
Vol. 12, No. 6, 1992
Newsletter
18. Italian Multi-centre Study: Epidemiology, clinical features and prognostic factors of pediatric HIV infection: Lancet 2:104_31046, 1988. 19. Johnson JP, Nair P, Hines SE, et al: Natural history and serologic diagnosis of infants born to human immunodeficiency virus infected women. Am J Dis Child 143:11471153, 1989. 20. Jones DS, Byers RH Bush TJ, et al.: The epidemiology of transfusion associated AIDS in children in the United States, 1989. Pediatrics. 21. Joshi V, Oleske J, Minnefor A, .et al.: Pathologic pulmonary findings in children with the acquired immunodeficiency syndrome: A study of ten cases. Human Pathol 16:241-246, 1985. 22. Marion RW, Wiznie AA, Hutcheon RG, et al.: Human T-cell lympholxophic virus type HI (HTLV-HI) embryopathy. Am J Dis Child 1986; 140:638-640. 23. Maur W. Potts B J, Robson AB: HIV-infection of first trimester and term human placerttal tissue: A possible mode of maternal-fetal transmission. J Infec Dis 160:583-588, 1989. 24. Mendez H. Landesman S Berthoud M, et al.: Natural history of infants bom to HIV1 seropositive mothers and their seronegarive controls. In: International Conference of the Implications of AIDS for Mothers and Infants. Paris, 1989. [Abstract C-41. 25. Mendez H: Natural history and prognostic factors. In: Yogev R, Connor E (ed); Management of HIV Infection of Infants and Children. Mosby Yearbook, 89-105, 1992. 26. Minkoff HL, Henderson C, Mendez H, et al.: Pregnancy outcomes among mothers infected with human immunodeficiency virus and uninfected controls. Am J Obstet/Gynecol 1990.
27. Mintz M, Epstein L, KoenigsbergerM: Neurologic manifestations of acquired hnmunodeficiency syndrome in children. Int Pediatr 4:161-171, 1989. 28. Oieske J: Natural history of HIV infection. In: Report of the Surgeon General's Workshop of Children with HIV Infection and Their Families, Washington, DC: DHHS: 24-25, 1987; DHHS Publication NO.(HRS)-D-MC87-1. 29. Oleske J, Mirmefor A, Cooper R, et al.: hnmunodeficiency syndrome in children. JAMA 249:2345-2349, 1983. 30. Oxtoby M: Perinatally acquired human hnmunodeficiency virus infection. Pediatr Infect Dis J 9:609-619, 1990. 31. Pahwa S, Keplan M, Fikrig S, et al: spectrum of HTLV-111 infection in children. JAMA 0255:2299-2305, 1986. 32. Pahwa S: Human hmnunodeficiency virus infection in children: Nature of ilmnunodeficiency clinical spectrum and management. Pediatr Infect Dis J 7:67-71, 1988. 33. Parks WP, Hutto C, Scott G: Pediatric AIDS: Prospects for prevention. In: Pappas T (ed): Gene Regulation and AIDS. Woodlands, Texas, Portfolio Publishing Co, pp. 247-253, 1990. 34. Rogers MF, Thomas PA, Slarcher ET, ct al: Acquired hnmunodeficiency syndrome in children: Report of the Centers for Disease Control National Surveillance; 1982 to 1985. Pediatrics 79:1008-1014, 1987. 35. Scott GB, Hutto C, Makuch R, el al.: Survival in children with perinatally acquired hmnan inununodeficiency virus type I infection. N Engl J Med 321:1791-1796, 1989. 36. Scott G, Mastrucci M: Puhnonary complications of HIV I infection in children. In: Yogev R, Cormor E (ects): Management of HIV Infection in Infants and Children. St. l_xmis, Mosby Yearbook, pp 323--355,
1992. 37. Semprini AE, Vucetich A, Pardi G, el al: HIV infection and AIDS in newborn babies of mothers positive for HIV antibody. Br Med J 294:610, 1987. 38. Stietun Er, Vink P: Transmission of human hmnunodeficiency virus, hffection by breast feeding. J Pediatr 118:410-412, 1991. 39. St Louis ME, Conway GA, Hayman CR, et al: Human i~mnunodeficiency virus infection in disadvantaged adolescents. Findings from the US Job Corps. JAMA 266: 23872391, 1991. 40. Sprcecher S, SomnenkoffG, Puissant F, cl al.: Vertical transmission of HIV in 15 week Ictus (letter). Lancet 2:288-289, 1985. 41. The European Collaborative Study. Mother to child transmission of HIV infection Lancet 2:1039-1043, 1988. 42. Thiry L, Sprecher-Goldbreechcr S, Jonckear T, et al: Isolation of AIDS virus from cell free breast milk of three healthy virus carriers. Lancet 1985: 981-892. 43. Van de Perr P, Shnonon A, Msellati P, et al: Postnatal transmission of human fimnunodeficiency virus type I from mother to infant. N Engl J Med 325:59344. Vovaisas E, Koch MA, Schifer A, et al.: LAV/HTLVHI in a 20 week fetus: (letter) Lancet 2:1129, 1985 45. Weinbreck P, Louslaud V, Denis F, ct al: Postuatal transmission of HIV infection Lancet 1:482, 1988. 46. Welter JH, Vaughan RD, Cohell AT: Psychosocial influences on acquired ilmnunodeficiency syndrome risk behaviors among high school students. Pediatrics: 88, 4:846, 1991. 47. Zigler JB, Cooper DA, Johnson RO, et al.: Postnatal transmission of AIDS-associated retrovirus from mother to infant. Lancet 1:89(~898, 1985.
Perinatai HIV Infection: Diagnostic Issues
directly. Because we now have reasonable alternatives for antiviral therapy and prophylaxis against opportunistic infections, the need for rapid and definitive diagnosis o f H I V is more imperative than ever.
bodies in a highly sensitive and specific manner. Enzyme immunoassay is a good, relatively inexpensive screening assay thal has an unacceptably high false positivity rate when used alone, especially in low risk populations. Confirming repeatedly reactive EIAs by Western blot has proven to be a very effective diagnostic approach. Two other assays for the general detection of HIV antibodies are indirect immuno fluorescence (IFA) and cadioimmunoprecipitation (RIPA). The use of Ihese assays
continuedfrom page 81
In the past few years, other diagnostic assays have been developed and/or applied to the problem of newborn/infant detection of HIV infection with some success. These are discussed below and have been separated into two groups based on whether they detect 1) the human immune response to HIV, or 2) the virus or its components
019%1859/92/$0.00 + 3.00
Assays Based on the Human (Immune) Response to HIV The most widely used assay for the detection of HIV infection is the E I A combined with confirmation by Western blot. Together, these two assays detect HIV anti-
© 1992 Elsevier Science Publishing Co., Inc.
!
V~I. 12, No. 6, 1992
in the past has been largely confined to research laboratories and has been generally supplanted by EIA/Western blot. Limitations for all these antibody assays occur very early in infection when the immune response has not yet been initiated (window period) and possibly late in the disease process when the immune system has been irreparably damaged. In addition, standard EIA and Western blot performed on at-risk babies cannot differentiate between antibodies derived from mother and infant, which poses the ultimate problem in early pediatric diagnosis. Because maternally acquired HIV antibodies are detectable in infants for an average of 12 months (and up to 18 to 21 months in the extreme), IgG-based antibody detection assays are not useful in these age groups. Since both IgM and IgA antibodies do not cross the placenta, the detection of HIV-specific antibodies of these isotypes has been postulated as the answer to HIV diagnosis in the newborn and young infant. This has not been an easy matter to accomplish, however, as has been observed from experience with other congenital infections. Problems with the detection of IgM antibodies to HIV include 1) the lack of an IgM response to HIV in some newborns and infants, 2) the short interval of time during which specific IgM is produced, and 3) interference in IgM detection assays by high levels of IgG and/or rheumatoid factor. Despite these limitations, several laboratories continue to pursue the detection of HIV-specific IgM as an answer to early diagnosis. The detection of HIV-specific IgA antibodies has been somewhat more rewarding and has been applied to a greater number of infants than IgM testing. The IgA adaptation of the EIA or Western blot detects infection in the vast majority of infants over the age of 6 months, and is reasonable in the 3 to 6 month age group, with rates of detection ranging from 57 to 96% in several reports. 9.~2.~5'It has been disappointing in studies to date, however, that have focused on infants below 3 months of age. Here, less than 50% of infected babies are detected, with sensitivity decreasing with decreasing age. IgA-based assays are currently in development and may ultimately prove to be useful diagnostic assays for the 6 to 24 month age group.
CLINICAL IMMUNOLOGY
Another promising assay focusing on the immune response is the detection of antibody secreting cells. This methodology has been termed ELISPOT (enzyme-linked immunospot) or IVAP (in vitro antibody production) and is characterized by the culture of isolated peripheral blood lymphocytes stimulated by HIV antigen either bound to a solid phase (nitrocellulose) or in solution. TM Previously sensitized lymphocytes (from an infected patient) will secrete HIV-specific antibodies that can then be detected in an EIA format. Small numbers of young infants have been studied to
B e c a u s e we now have reasonable alternatives for antiviral therapy and prophylaxis against opportunistic infections, the need for rapid and definitive diagnosis of HIV is more imperative than ever.
date, but preliminary results appear encouraging with detection of HIV antibody secretion in 60 to 80% of infants studied. TM The use of flow cytometry to enumerate T-lymphocyte subsets has become a universal tool in the evaluation of an individual at risk for or known to be infected with HIV. The quantification of T-helper lymphocytes (CD4 lymphocytes) has proven to be the most useful marker of disease activity and clinical course. Although the CD4 lymphocyte count is not a useful diagnostic parameter, it is used routinely in both children and adults when evaluating the need for antiviral therapy and prophylaxis against opportunistic infections, most notably Pneumocytiscarinii.2It must be emphasized that normal CD4 values established for adults are not appropriate for children, the latter having significantly higher cell numbers. :,4 Assays Based on Detection of the Virus or Its Components
The direct detection of HIV has significant © 1992 Elsevier Science Publishing Co., Inc.
Nelvsletter
87
theoretical advantages for diagnostic application in young infants. Specifically, this approach does not rely on an effective human immune response with the associated problems of passively acquired maternal antibody and the suboptimal capacity of the developing newborn immune system. However, these techniques, to be discussed below, generally require more technical sophistication and expense than EIA-based assays. The standard virologic technique for virus identification in clinical specimens is virus culture. Purified peripheral blood mononuclear cells (PBLs) from the test subject are cocultured with uninfected, stimulated PBLs from a normal donor. The cells are incubated for a period of 3 to 4 weeks, during which time aliquots of the culture media are removed periodically. These culture aliquots are then assayed for HIV p24 antigen or reverse tmnscriptase as evidence of viral replication in the culture system. Most laboratories identify 90 to 100% of HIV-infected adults.7 Somewhat lower rates are achieved by for infected infants and pregnant women. However, below 3 months of age the rates decline significantly and are <50% in the first month of 1ife3'5'6'8Factors that may adversely affect the ability to detect virus by culture include low viral burden early in infection, sequestering of virus in noncirculating cell compartments, and the presence of large numbers of suppressor T lymphocytes. One technique for the detection of viral components is the p24 antigen assay, p24 antigen is a structural protein that composes the viral core and which is detectable in serum by an EIA assay. Commercially available assays are relatively insensitive and will usually detect p24 antigen for a brief interval early in infection and then later in infection when the production of viral proteins and particles increases. Therefore, this assay has not proven useful in a diagnostic sense, but has some application for evaluation of an individual's clinical course and response to therapy. One reason for the relative insensitivity of the p24 antigen assay is that it complexes with excess antibody present in the circulation. These antibody-antigen com0197-1859/92/$0.00 + 3.00
88 ' C L I N I C A L I M M U N O L O G Y Newsletter
plexes are not detectable by the standard assay. Methods to dissociate complexes prior to assay (e.g., acid hydrolysis) have been employed and appear quite promising. Sensitivity increases dramatically, with p24 antigen becoming detectable in a majority of individuals, including infants. Whether this new approach will find a niche in our diagnostic armamentarium remains to be ascertained. A concern arising from preliminary studies in newborns is that of false positives. It appears that p24 antigen may cross the placenta from mother to fetus and be detectable for days to weeks in children ultimately proven to be uninfected. Further studies are clearly warranted to define the role of this assay in early childhood diagnosis. A promising new assay that utilizes molecular genetic techniques for HIV identification is the polymerase chain reaction (I:'CR). This approach allows the identification of HIV DNA present in infected cells. A target HIV gene or gene fragment is copied millions of times by means of an enzymatic reaction, ~ffter which the molecules are easily detected. The technique is extremely sensitive in that even a single infected cell can theoretically be identified. This high degree of sensitivity has also been an Achilles' heel, in that even slight contamination from specimen to specimen at the molecular level will result in falsepositive assays. Much effort has been devoted to preventing contamination problems, focusing on meticulous technique and "laboratory tricks." Even so, extensive panels of positive and negative controls need to be incorporated with each batch of PCR assays. Many studies have documented close to 100% sensitivity and specificity of PCR in HIV-infected adults and children above 3 months of age. v* Between birth and 3 months of age, sensitivity falls somewhat but is probably higher than that seen for culture. 3,5,6,8,1°:3As with the other assays mentioned above, relatively few young infants have been studied whose ultimate infection status is known. Two studies published by Comeau et al? and Rogers et alJ 3 reported a combined PCR detection rate in the neonatal period (birth to 4 weeks) of 57% (8/14). Sensitivity rapidly improves in the second and third months of life, at which time virtually every in0197-1859/92/$0.00 + 3.00
Vol. 12, No. 6, i992
fected infant can be detected. Currently, PCR is primarily a research tool, although it is available at several commercial laboratories. PCR technology in kit format is rapidly being developed and may be available in the near future. It is anticipated to be quite user friendly and will provide same day or next day results, a vast improvement over the 3 to 4 week turnaround time required by culture. Utilization of PCR for clinical diagnosis still will be required to pass the hurdles of F.D.A. approval and widespread clinical testing, especially for application to diagnosis of young infants.
Summary HIV diagnosis for the majority of at-risk individuals---children, adults, and pregnant women--can readily be accomplished by the standard approach of EIA and Western blot. Children under 18 to 24 months of age pose a special diagnostic problem due to the presence of passively acquired, maternal antibodies to HIV. It is becoming imperative that definitive diagnosis of infants be accomplished as early in life as possible because of current or anticipated 1hempeutic options and prophylactic measures. Diagnostic approaches other than EIA/Westem blot need to be considered and/or developed for the young infant at risk for infection. The two most promising assays are PCR and viral culture. To date, very few newhorns and young infants have been evaluated by these assays as evidenced by the published scientific literature. Several pertnatal collaborative studies, both in the U.S. and abroad, hope to fill this knowledge gap in the near future. Almost all HIV-infected infants can be detected now by 3 months of age. It seems reasonable to expect that up to 25% of newborns will have undetectable infection utilizing any foreseeable technique. This subgroup apparently has very low levels of circulating virus, implying that HIV may reside in fixed tissue or privileged sites, such as the central nervous system. Although we have come a long way from what was state-ofthe-art practice just a few years ago, it is clear that further research is warranted concerning the pathogenesis and diagnosis of © 1992ElsevierScience Publishing Co., Inc.
HIV in the perinatal pericxl.
References 1. Amadori A, DcRossi A, Chieco-Bianchi L, et al.: Diagnosis of HIV-I infection in infants: in vitro production of virus-specific antibody in lymphocytes. Pediatr Infect Dis J 9:26-30, 1990. 2. Centers for Disease Control: Guidelines for prophylaxis against Pneumocystis carinii pneumonia for children infected with HIV. MMWR 40:RR-2, 1991. 3. Comeau AM, Harris J, Mclntosh K, et al.: I~R in detecting IV infection among seropositive infants: Relation to clinical status and age and to results of other assays. J AIDS 5:271-288, 1992. 4. Denny T, Yogev R, Gehnan R, et al.: Lym~ phocyte subsets in healthy children during the first 5 years of life. JAMA 26714841488, 1992. 5. Edwards JR, Ulrich PP, Weintrub PS, cl al.: PCR compared with concurrent viral cultures for rapid identification of HIV infection among high risk infants and children. J Pediatr 115:200--203, 1989. 6. Escaich S, Wallon M, Baginski 1, et al.: Comparison of HIV detection by virus isolation in lymphocyte cultures and molecular amplification of HIV DNA and RNA by PCR in offspring of seropositive mothers. J AIDS 4:130--135, 1991. 7. Jackson J, Kwok SY, Sninsky JJ, et al.: HIV type 1 detected in all seropositivc symptomatic and asymptomatic individuals. J Clin Microbiol 28:16-19, 1990. Krivine A, Yakudhna A, LeMay M, el al.: A comparative study of virus isolation, DCR, and antigen detection in children of mothers infected with HIV. J F'cdiatr 116:372-376, 1990. 9. 'Landsman S, Weiblen B, Mendez H, et al.: Clinical utility of HIV-IgA hmnunoblot assay in the early diagnosis of perinatal HIV infeclion. JAMA 266:3443-3446, 1991. 10. Laure F, Courgnaud V, Rouzioux C, et al.: Detection of HIV-I DNA ha infants and children by means of the I:~R Lancet 2:538-541, 1988. 11. Lee FK, Natunias AJ, Lowery S, et al.: ELISFgOT: A new approach to studying the dynamics of virus-inunune system interaction for diagnosis and monitoring of HIV infection. AIDS Res Hum Retroviruscs 5:517--523, 1989. 12. Quinn TC, Kline RL, Halsey N, ctal.: Early diagnosis of perinatal HIV infeclion i~y detection of virat-~,pecific lgA
C L I N I C A L I M M U N O L O G Y Newsletter 89
Vol. 12, No. 6, 1992
antibodies. JAMA 266:3439-3442, 1991. 13. Roger's MF, Ou Cy, Rayfield M, et al.: Use of PCR for early detection of the proviral
sequencesof HIV in infantsborn to seropositivemollaers.N Eng J Med 320:1649-
Treatment
1654, 1989. 14. Sheppard HW, Ascher MS, Musch MP, et al. A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1. J Acquit Immune Defic Syndr
of HIV Infection
In Infants
4:277-283, 1991. 15. Weiblen BJ, Lee FK, Cooper ER, et al.: Early diagnosis of HIV infection in infants by detection oflgA antibodies. Lancet 335:988-990, 1990.
and Children
Samuel Grubman and Edward Connor Department of Pediatrics, National Pediatric HIV Resource Center, AIDS Clinical Trials Unit, UMD-New Jersey Medical School, Children's Hospital of New Jersey, Newark, New Jersey
he medical management of human immunodeficiency virus (HIV) infection in infants and children is multifaceted. HIV affects almost every aspect of an infected child's health and effective therapy must be directed at both the virus and the disease manifestations caused by the viral infection. Standard treatment regimens, therefore, include antiretroviral therapy, specific antimicrobial prophylaxis and treatment of opportunistic infections, immunomodulatory therapy, and nutritional support, as well as anticipatory management of routine childhood illnesses and comprehensive well child care.
T
Pathogenesis An understanding of the pathogenesis of H1V infection is essential to making rational therapeutic decisions, especially those related to antiretroviral treatment. HIV is an enveloped single-stranded RNA virus and a member of the lentivirus group of retroviruses. These viruses characteristically have a prolonged incubation period, tropism for hematopoietic and nervous system tissues, and immune-altering effects. Attachment of HIV to target host cells occurs primarily via interaction of viral envelope gpl20 with the CD4 receptor molecule on host cells. Evidence suggests that HIV not only infects CIM+ lymphocytes and macrophages, but also bone marrow precursor cells, thymic cells and some cells of end organ tissues, such as the central nervous system and gastrointestinal tract. Following entry into target cells, DNA is synthesized from viral RNA by reverse transcriptase, and this DNA is then integrated into the host cell genome,
where it remains as a provirus. Upon cellular activation, HIV replication occurs and new viral particles are produced that are capable of infecting new cells. HIV- 1 reverse transcriptase is, however, inefficient, and HIV replication frequently results in mutations that may give rise to heterogenous viral particles? Early in HIV infection, presentation of viral antigens to the host's immune system produces an effective and coordinated cellmediated and humoral immune response, with the generation of CD4+ T lymphocytes, B lymphocytes, and CD8+ cytotoxic T lymphocytes targeted against HIV. 2"3 This early response is usually effective in the control, but not the elimination, of HIV infection. After the primary immune response, a period of latent infection begins, with "pure" latency consisting of the integration of proviral DNA in the infected cell's nucleus, but without active viral replication or presentation of viral proteins on the cell surface. It is thought that this stage coincides with the prolonged incubation period seen in adults and older children with HIV infection. Activation of the immune system, though, as occurs with intercurrent infection, leads to intermittent viral replication, and latency is actually characterized by periods of viral replication alternating with true latent infection.2 With continued viral replication, the host's immune response is eventually "overwhelmed" and rendered ineffective in controlling the virus. One reason for this may be the propensity for viral heterogeneity with the development of alterations in viral RNA and the resultant establishment in each infected individual © 1992 Elsevier Science Publishing Co., Inc.
of a mixture of viral strains with altered immunogenicity, cytopathogenicity and cell tropism.2.4 Viral strains that are less immunogenic, lacking the epitopes recognized by the antibody- and cell-mediated immune response established during initial infection, can evade the immune response and may preferentially survive. In addition, the cytolytic affects of HIV on CD4+ lymphocytes and other immune alterations that occur as a results of HIV infection cause a state of immunodeficiency, resulting in impaired immune responses to HIV and other infectious agents. During the early stages of HIV infection, which usually manifests clinically as asymptomatic infection, there appears to be an intricate balance between viral replications and host immune response. As the disease progresses, attrition of the immune system occurs, ultimately associated with a fall in CD4+ iymphocytes. It is known that most CD4+ lymphocytes in the circulation during the asymptomatic period are not infected with HIV. The exact mechanism for depletion of infected and uninfected CD4+ lymphocytes is unknown but probably results from a combination of 1) immune mechanisms directed against infected cells, 2) direct viral damage to infected cells, 3) antibody-dependent cellular cytotoxicity against uninfected gp 120-coated cells, 4) autoimmune responses against gpl20 and gp41 homologous epitopes, 5) formation of syncytia, and 6) chronic partial CD4+ lymphocyte activation. ~'3 As immune attrition continues, the balance shifts in favor of HIV and there is a resultant increase in both replication of virus and percentage of circulating 0197-1859/92/$0.00 + 3.00
Vol. 12, No. 6, 1992
Newsletter
18. Italian Multi-centre Study: Epidemiology, clinical features and prognostic factors of pediatric HIV infection: Lancet 2:104_31046, 1988. 19. Johnson JP, Nair P, Hines SE, et al: Natural history and serologic diagnosis of infants born to human immunodeficiency virus infected women. Am J Dis Child 143:11471153, 1989. 20. Jones DS, Byers RH Bush TJ, et al.: The epidemiology of transfusion associated AIDS in children in the United States, 1989. Pediatrics. 21. Joshi V, Oleske J, Minnefor A, .et al.: Pathologic pulmonary findings in children with the acquired immunodeficiency syndrome: A study of ten cases. Human Pathol 16:241-246, 1985. 22. Marion RW, Wiznie AA, Hutcheon RG, et al.: Human T-cell lympholxophic virus type HI (HTLV-HI) embryopathy. Am J Dis Child 1986; 140:638-640. 23. Maur W. Potts B J, Robson AB: HIV-infection of first trimester and term human placerttal tissue: A possible mode of maternal-fetal transmission. J Infec Dis 160:583-588, 1989. 24. Mendez H. Landesman S Berthoud M, et al.: Natural history of infants bom to HIV1 seropositive mothers and their seronegarive controls. In: International Conference of the Implications of AIDS for Mothers and Infants. Paris, 1989. [Abstract C-41. 25. Mendez H: Natural history and prognostic factors. In: Yogev R, Connor E (ed); Management of HIV Infection of Infants and Children. Mosby Yearbook, 89-105, 1992. 26. Minkoff HL, Henderson C, Mendez H, et al.: Pregnancy outcomes among mothers infected with human immunodeficiency virus and uninfected controls. Am J Obstet/Gynecol 1990.
27. Mintz M, Epstein L, KoenigsbergerM: Neurologic manifestations of acquired hnmunodeficiency syndrome in children. Int Pediatr 4:161-171, 1989. 28. Oieske J: Natural history of HIV infection. In: Report of the Surgeon General's Workshop of Children with HIV Infection and Their Families, Washington, DC: DHHS: 24-25, 1987; DHHS Publication NO.(HRS)-D-MC87-1. 29. Oleske J, Mirmefor A, Cooper R, et al.: hnmunodeficiency syndrome in children. JAMA 249:2345-2349, 1983. 30. Oxtoby M: Perinatally acquired human hnmunodeficiency virus infection. Pediatr Infect Dis J 9:609-619, 1990. 31. Pahwa S, Keplan M, Fikrig S, et al: spectrum of HTLV-111 infection in children. JAMA 0255:2299-2305, 1986. 32. Pahwa S: Human hmnunodeficiency virus infection in children: Nature of ilmnunodeficiency clinical spectrum and management. Pediatr Infect Dis J 7:67-71, 1988. 33. Parks WP, Hutto C, Scott G: Pediatric AIDS: Prospects for prevention. In: Pappas T (ed): Gene Regulation and AIDS. Woodlands, Texas, Portfolio Publishing Co, pp. 247-253, 1990. 34. Rogers MF, Thomas PA, Slarcher ET, ct al: Acquired hnmunodeficiency syndrome in children: Report of the Centers for Disease Control National Surveillance; 1982 to 1985. Pediatrics 79:1008-1014, 1987. 35. Scott GB, Hutto C, Makuch R, el al.: Survival in children with perinatally acquired hmnan inununodeficiency virus type I infection. N Engl J Med 321:1791-1796, 1989. 36. Scott G, Mastrucci M: Puhnonary complications of HIV I infection in children. In: Yogev R, Cormor E (ects): Management of HIV Infection in Infants and Children. St. l_xmis, Mosby Yearbook, pp 323--355,
1992. 37. Semprini AE, Vucetich A, Pardi G, el al: HIV infection and AIDS in newborn babies of mothers positive for HIV antibody. Br Med J 294:610, 1987. 38. Stietun Er, Vink P: Transmission of human hmnunodeficiency virus, hffection by breast feeding. J Pediatr 118:410-412, 1991. 39. St Louis ME, Conway GA, Hayman CR, et al: Human i~mnunodeficiency virus infection in disadvantaged adolescents. Findings from the US Job Corps. JAMA 266: 23872391, 1991. 40. Sprcecher S, SomnenkoffG, Puissant F, cl al.: Vertical transmission of HIV in 15 week Ictus (letter). Lancet 2:288-289, 1985. 41. The European Collaborative Study. Mother to child transmission of HIV infection Lancet 2:1039-1043, 1988. 42. Thiry L, Sprecher-Goldbreechcr S, Jonckear T, et al: Isolation of AIDS virus from cell free breast milk of three healthy virus carriers. Lancet 1985: 981-892. 43. Van de Perr P, Shnonon A, Msellati P, et al: Postnatal transmission of human fimnunodeficiency virus type I from mother to infant. N Engl J Med 325:59344. Vovaisas E, Koch MA, Schifer A, et al.: LAV/HTLVHI in a 20 week fetus: (letter) Lancet 2:1129, 1985 45. Weinbreck P, Louslaud V, Denis F, ct al: Postuatal transmission of HIV infection Lancet 1:482, 1988. 46. Welter JH, Vaughan RD, Cohell AT: Psychosocial influences on acquired ilmnunodeficiency syndrome risk behaviors among high school students. Pediatrics: 88, 4:846, 1991. 47. Zigler JB, Cooper DA, Johnson RO, et al.: Postnatal transmission of AIDS-associated retrovirus from mother to infant. Lancet 1:89(~898, 1985.
Perinatai HIV Infection: Diagnostic Issues
directly. Because we now have reasonable alternatives for antiviral therapy and prophylaxis against opportunistic infections, the need for rapid and definitive diagnosis o f H I V is more imperative than ever.
bodies in a highly sensitive and specific manner. Enzyme immunoassay is a good, relatively inexpensive screening assay thal has an unacceptably high false positivity rate when used alone, especially in low risk populations. Confirming repeatedly reactive EIAs by Western blot has proven to be a very effective diagnostic approach. Two other assays for the general detection of HIV antibodies are indirect immuno fluorescence (IFA) and cadioimmunoprecipitation (RIPA). The use of Ihese assays
continuedfrom page 81
In the past few years, other diagnostic assays have been developed and/or applied to the problem of newborn/infant detection of HIV infection with some success. These are discussed below and have been separated into two groups based on whether they detect 1) the human immune response to HIV, or 2) the virus or its components
019%1859/92/$0.00 + 3.00
Assays Based on the Human (Immune) Response to HIV The most widely used assay for the detection of HIV infection is the E I A combined with confirmation by Western blot. Together, these two assays detect HIV anti-
© 1992 Elsevier Science Publishing Co., Inc.
!
V~I. 12, No. 6, 1992
in the past has been largely confined to research laboratories and has been generally supplanted by EIA/Western blot. Limitations for all these antibody assays occur very early in infection when the immune response has not yet been initiated (window period) and possibly late in the disease process when the immune system has been irreparably damaged. In addition, standard EIA and Western blot performed on at-risk babies cannot differentiate between antibodies derived from mother and infant, which poses the ultimate problem in early pediatric diagnosis. Because maternally acquired HIV antibodies are detectable in infants for an average of 12 months (and up to 18 to 21 months in the extreme), IgG-based antibody detection assays are not useful in these age groups. Since both IgM and IgA antibodies do not cross the placenta, the detection of HIV-specific antibodies of these isotypes has been postulated as the answer to HIV diagnosis in the newborn and young infant. This has not been an easy matter to accomplish, however, as has been observed from experience with other congenital infections. Problems with the detection of IgM antibodies to HIV include 1) the lack of an IgM response to HIV in some newborns and infants, 2) the short interval of time during which specific IgM is produced, and 3) interference in IgM detection assays by high levels of IgG and/or rheumatoid factor. Despite these limitations, several laboratories continue to pursue the detection of HIV-specific IgM as an answer to early diagnosis. The detection of HIV-specific IgA antibodies has been somewhat more rewarding and has been applied to a greater number of infants than IgM testing. The IgA adaptation of the EIA or Western blot detects infection in the vast majority of infants over the age of 6 months, and is reasonable in the 3 to 6 month age group, with rates of detection ranging from 57 to 96% in several reports. 9.~2.~5'It has been disappointing in studies to date, however, that have focused on infants below 3 months of age. Here, less than 50% of infected babies are detected, with sensitivity decreasing with decreasing age. IgA-based assays are currently in development and may ultimately prove to be useful diagnostic assays for the 6 to 24 month age group.
CLINICAL IMMUNOLOGY
Another promising assay focusing on the immune response is the detection of antibody secreting cells. This methodology has been termed ELISPOT (enzyme-linked immunospot) or IVAP (in vitro antibody production) and is characterized by the culture of isolated peripheral blood lymphocytes stimulated by HIV antigen either bound to a solid phase (nitrocellulose) or in solution. TM Previously sensitized lymphocytes (from an infected patient) will secrete HIV-specific antibodies that can then be detected in an EIA format. Small numbers of young infants have been studied to
B e c a u s e we now have reasonable alternatives for antiviral therapy and prophylaxis against opportunistic infections, the need for rapid and definitive diagnosis of HIV is more imperative than ever.
date, but preliminary results appear encouraging with detection of HIV antibody secretion in 60 to 80% of infants studied. TM The use of flow cytometry to enumerate T-lymphocyte subsets has become a universal tool in the evaluation of an individual at risk for or known to be infected with HIV. The quantification of T-helper lymphocytes (CD4 lymphocytes) has proven to be the most useful marker of disease activity and clinical course. Although the CD4 lymphocyte count is not a useful diagnostic parameter, it is used routinely in both children and adults when evaluating the need for antiviral therapy and prophylaxis against opportunistic infections, most notably Pneumocytiscarinii.2It must be emphasized that normal CD4 values established for adults are not appropriate for children, the latter having significantly higher cell numbers. :,4 Assays Based on Detection of the Virus or Its Components
The direct detection of HIV has significant © 1992 Elsevier Science Publishing Co., Inc.
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87
theoretical advantages for diagnostic application in young infants. Specifically, this approach does not rely on an effective human immune response with the associated problems of passively acquired maternal antibody and the suboptimal capacity of the developing newborn immune system. However, these techniques, to be discussed below, generally require more technical sophistication and expense than EIA-based assays. The standard virologic technique for virus identification in clinical specimens is virus culture. Purified peripheral blood mononuclear cells (PBLs) from the test subject are cocultured with uninfected, stimulated PBLs from a normal donor. The cells are incubated for a period of 3 to 4 weeks, during which time aliquots of the culture media are removed periodically. These culture aliquots are then assayed for HIV p24 antigen or reverse tmnscriptase as evidence of viral replication in the culture system. Most laboratories identify 90 to 100% of HIV-infected adults.7 Somewhat lower rates are achieved by for infected infants and pregnant women. However, below 3 months of age the rates decline significantly and are <50% in the first month of 1ife3'5'6'8Factors that may adversely affect the ability to detect virus by culture include low viral burden early in infection, sequestering of virus in noncirculating cell compartments, and the presence of large numbers of suppressor T lymphocytes. One technique for the detection of viral components is the p24 antigen assay, p24 antigen is a structural protein that composes the viral core and which is detectable in serum by an EIA assay. Commercially available assays are relatively insensitive and will usually detect p24 antigen for a brief interval early in infection and then later in infection when the production of viral proteins and particles increases. Therefore, this assay has not proven useful in a diagnostic sense, but has some application for evaluation of an individual's clinical course and response to therapy. One reason for the relative insensitivity of the p24 antigen assay is that it complexes with excess antibody present in the circulation. These antibody-antigen com0197-1859/92/$0.00 + 3.00
88 ' C L I N I C A L I M M U N O L O G Y Newsletter
plexes are not detectable by the standard assay. Methods to dissociate complexes prior to assay (e.g., acid hydrolysis) have been employed and appear quite promising. Sensitivity increases dramatically, with p24 antigen becoming detectable in a majority of individuals, including infants. Whether this new approach will find a niche in our diagnostic armamentarium remains to be ascertained. A concern arising from preliminary studies in newborns is that of false positives. It appears that p24 antigen may cross the placenta from mother to fetus and be detectable for days to weeks in children ultimately proven to be uninfected. Further studies are clearly warranted to define the role of this assay in early childhood diagnosis. A promising new assay that utilizes molecular genetic techniques for HIV identification is the polymerase chain reaction (I:'CR). This approach allows the identification of HIV DNA present in infected cells. A target HIV gene or gene fragment is copied millions of times by means of an enzymatic reaction, ~ffter which the molecules are easily detected. The technique is extremely sensitive in that even a single infected cell can theoretically be identified. This high degree of sensitivity has also been an Achilles' heel, in that even slight contamination from specimen to specimen at the molecular level will result in falsepositive assays. Much effort has been devoted to preventing contamination problems, focusing on meticulous technique and "laboratory tricks." Even so, extensive panels of positive and negative controls need to be incorporated with each batch of PCR assays. Many studies have documented close to 100% sensitivity and specificity of PCR in HIV-infected adults and children above 3 months of age. v* Between birth and 3 months of age, sensitivity falls somewhat but is probably higher than that seen for culture. 3,5,6,8,1°:3As with the other assays mentioned above, relatively few young infants have been studied whose ultimate infection status is known. Two studies published by Comeau et al? and Rogers et alJ 3 reported a combined PCR detection rate in the neonatal period (birth to 4 weeks) of 57% (8/14). Sensitivity rapidly improves in the second and third months of life, at which time virtually every in0197-1859/92/$0.00 + 3.00
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fected infant can be detected. Currently, PCR is primarily a research tool, although it is available at several commercial laboratories. PCR technology in kit format is rapidly being developed and may be available in the near future. It is anticipated to be quite user friendly and will provide same day or next day results, a vast improvement over the 3 to 4 week turnaround time required by culture. Utilization of PCR for clinical diagnosis still will be required to pass the hurdles of F.D.A. approval and widespread clinical testing, especially for application to diagnosis of young infants.
Summary HIV diagnosis for the majority of at-risk individuals---children, adults, and pregnant women--can readily be accomplished by the standard approach of EIA and Western blot. Children under 18 to 24 months of age pose a special diagnostic problem due to the presence of passively acquired, maternal antibodies to HIV. It is becoming imperative that definitive diagnosis of infants be accomplished as early in life as possible because of current or anticipated 1hempeutic options and prophylactic measures. Diagnostic approaches other than EIA/Westem blot need to be considered and/or developed for the young infant at risk for infection. The two most promising assays are PCR and viral culture. To date, very few newhorns and young infants have been evaluated by these assays as evidenced by the published scientific literature. Several pertnatal collaborative studies, both in the U.S. and abroad, hope to fill this knowledge gap in the near future. Almost all HIV-infected infants can be detected now by 3 months of age. It seems reasonable to expect that up to 25% of newborns will have undetectable infection utilizing any foreseeable technique. This subgroup apparently has very low levels of circulating virus, implying that HIV may reside in fixed tissue or privileged sites, such as the central nervous system. Although we have come a long way from what was state-ofthe-art practice just a few years ago, it is clear that further research is warranted concerning the pathogenesis and diagnosis of © 1992ElsevierScience Publishing Co., Inc.
HIV in the perinatal pericxl.
References 1. Amadori A, DcRossi A, Chieco-Bianchi L, et al.: Diagnosis of HIV-I infection in infants: in vitro production of virus-specific antibody in lymphocytes. Pediatr Infect Dis J 9:26-30, 1990. 2. Centers for Disease Control: Guidelines for prophylaxis against Pneumocystis carinii pneumonia for children infected with HIV. MMWR 40:RR-2, 1991. 3. Comeau AM, Harris J, Mclntosh K, et al.: I~R in detecting IV infection among seropositive infants: Relation to clinical status and age and to results of other assays. J AIDS 5:271-288, 1992. 4. Denny T, Yogev R, Gehnan R, et al.: Lym~ phocyte subsets in healthy children during the first 5 years of life. JAMA 26714841488, 1992. 5. Edwards JR, Ulrich PP, Weintrub PS, cl al.: PCR compared with concurrent viral cultures for rapid identification of HIV infection among high risk infants and children. J Pediatr 115:200--203, 1989. 6. Escaich S, Wallon M, Baginski 1, et al.: Comparison of HIV detection by virus isolation in lymphocyte cultures and molecular amplification of HIV DNA and RNA by PCR in offspring of seropositive mothers. J AIDS 4:130--135, 1991. 7. Jackson J, Kwok SY, Sninsky JJ, et al.: HIV type 1 detected in all seropositivc symptomatic and asymptomatic individuals. J Clin Microbiol 28:16-19, 1990. Krivine A, Yakudhna A, LeMay M, el al.: A comparative study of virus isolation, DCR, and antigen detection in children of mothers infected with HIV. J F'cdiatr 116:372-376, 1990. 9. 'Landsman S, Weiblen B, Mendez H, et al.: Clinical utility of HIV-IgA hmnunoblot assay in the early diagnosis of perinatal HIV infeclion. JAMA 266:3443-3446, 1991. 10. Laure F, Courgnaud V, Rouzioux C, et al.: Detection of HIV-I DNA ha infants and children by means of the I:~R Lancet 2:538-541, 1988. 11. Lee FK, Natunias AJ, Lowery S, et al.: ELISFgOT: A new approach to studying the dynamics of virus-inunune system interaction for diagnosis and monitoring of HIV infection. AIDS Res Hum Retroviruscs 5:517--523, 1989. 12. Quinn TC, Kline RL, Halsey N, ctal.: Early diagnosis of perinatal HIV infeclion i~y detection of virat-~,pecific lgA
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antibodies. JAMA 266:3439-3442, 1991. 13. Roger's MF, Ou Cy, Rayfield M, et al.: Use of PCR for early detection of the proviral
sequencesof HIV in infantsborn to seropositivemollaers.N Eng J Med 320:1649-
Treatment
1654, 1989. 14. Sheppard HW, Ascher MS, Musch MP, et al. A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1. J Acquit Immune Defic Syndr
of HIV Infection
In Infants
4:277-283, 1991. 15. Weiblen BJ, Lee FK, Cooper ER, et al.: Early diagnosis of HIV infection in infants by detection oflgA antibodies. Lancet 335:988-990, 1990.
and Children
Samuel Grubman and Edward Connor Department of Pediatrics, National Pediatric HIV Resource Center, AIDS Clinical Trials Unit, UMD-New Jersey Medical School, Children's Hospital of New Jersey, Newark, New Jersey
he medical management of human immunodeficiency virus (HIV) infection in infants and children is multifaceted. HIV affects almost every aspect of an infected child's health and effective therapy must be directed at both the virus and the disease manifestations caused by the viral infection. Standard treatment regimens, therefore, include antiretroviral therapy, specific antimicrobial prophylaxis and treatment of opportunistic infections, immunomodulatory therapy, and nutritional support, as well as anticipatory management of routine childhood illnesses and comprehensive well child care.
T
Pathogenesis An understanding of the pathogenesis of H1V infection is essential to making rational therapeutic decisions, especially those related to antiretroviral treatment. HIV is an enveloped single-stranded RNA virus and a member of the lentivirus group of retroviruses. These viruses characteristically have a prolonged incubation period, tropism for hematopoietic and nervous system tissues, and immune-altering effects. Attachment of HIV to target host cells occurs primarily via interaction of viral envelope gpl20 with the CD4 receptor molecule on host cells. Evidence suggests that HIV not only infects CIM+ lymphocytes and macrophages, but also bone marrow precursor cells, thymic cells and some cells of end organ tissues, such as the central nervous system and gastrointestinal tract. Following entry into target cells, DNA is synthesized from viral RNA by reverse transcriptase, and this DNA is then integrated into the host cell genome,
where it remains as a provirus. Upon cellular activation, HIV replication occurs and new viral particles are produced that are capable of infecting new cells. HIV- 1 reverse transcriptase is, however, inefficient, and HIV replication frequently results in mutations that may give rise to heterogenous viral particles? Early in HIV infection, presentation of viral antigens to the host's immune system produces an effective and coordinated cellmediated and humoral immune response, with the generation of CD4+ T lymphocytes, B lymphocytes, and CD8+ cytotoxic T lymphocytes targeted against HIV. 2"3 This early response is usually effective in the control, but not the elimination, of HIV infection. After the primary immune response, a period of latent infection begins, with "pure" latency consisting of the integration of proviral DNA in the infected cell's nucleus, but without active viral replication or presentation of viral proteins on the cell surface. It is thought that this stage coincides with the prolonged incubation period seen in adults and older children with HIV infection. Activation of the immune system, though, as occurs with intercurrent infection, leads to intermittent viral replication, and latency is actually characterized by periods of viral replication alternating with true latent infection.2 With continued viral replication, the host's immune response is eventually "overwhelmed" and rendered ineffective in controlling the virus. One reason for this may be the propensity for viral heterogeneity with the development of alterations in viral RNA and the resultant establishment in each infected individual © 1992 Elsevier Science Publishing Co., Inc.
of a mixture of viral strains with altered immunogenicity, cytopathogenicity and cell tropism.2.4 Viral strains that are less immunogenic, lacking the epitopes recognized by the antibody- and cell-mediated immune response established during initial infection, can evade the immune response and may preferentially survive. In addition, the cytolytic affects of HIV on CD4+ lymphocytes and other immune alterations that occur as a results of HIV infection cause a state of immunodeficiency, resulting in impaired immune responses to HIV and other infectious agents. During the early stages of HIV infection, which usually manifests clinically as asymptomatic infection, there appears to be an intricate balance between viral replications and host immune response. As the disease progresses, attrition of the immune system occurs, ultimately associated with a fall in CD4+ iymphocytes. It is known that most CD4+ lymphocytes in the circulation during the asymptomatic period are not infected with HIV. The exact mechanism for depletion of infected and uninfected CD4+ lymphocytes is unknown but probably results from a combination of 1) immune mechanisms directed against infected cells, 2) direct viral damage to infected cells, 3) antibody-dependent cellular cytotoxicity against uninfected gp 120-coated cells, 4) autoimmune responses against gpl20 and gp41 homologous epitopes, 5) formation of syncytia, and 6) chronic partial CD4+ lymphocyte activation. ~'3 As immune attrition continues, the balance shifts in favor of HIV and there is a resultant increase in both replication of virus and percentage of circulating 0197-1859/92/$0.00 + 3.00