Original Article
The Use of Viral Culture and p24 Antigen Testing to Diagnose Human Immunodeficiency Virus Infection in Neonates
N Engl J Med 1992; 327:1192-1197October 22, 1992
- Abstract
- Abstract
Background.
Early diagnosis of human immunodeficiency virus (HIV) infection in infants born to infected mothers is important for the infants' medical care, but the presence of maternal antibodies makes serologic tests uninformative.
Methods.
In a cohort study of 181 infants born to HIV-infected mothers, we assessed the diagnostic value of HIV viral culture and testing for the presence of p24 antigen. The infants were tested at birth, again during the first 3 months, then followed and tested at the age of at least 18 months.
Results.
Of the 181 infants, 3 died of HIV infection and 37 were seropositive after the age of 18 months. Viral cultures at birth were positive in 19 of the 40 infected infants and in none of the uninfected infants, yielding a sensitivity of 48 percent (95 percent confidence interval, 32 to 63 percent) and a specificity of 100 percent (95 percent confidence interval, 97 to 100 percent). By the age of three months, 30 of the 40 infants (75 percent) had positive cultures; again, there were no false positive results among the infants who were tested a second time, of the 141 who remained uninfected. The sensitivity of testing for p24 antigen at birth was only 18 percent, with a specificity of 100 percent. The presence of p24 antigen at birth was associated with the development of early and severe HIV-related disease (P<0.04).
Conclusions.
Viral culture at birth can correctly identify about half of newborns with HIV infection. The fact that this usually sensitive technique fails to identify about half the ultimately infected neonates suggests that vertical transmission of HIV may occur late in pregnancy or during delivery. (N Engl J Med 1992;327:1192–7.)
Media in This Article
Table 1
Characteristics of Mothers and Infants, According to Whether the Infant Underwent Viral Culture at Birth.*Table 2
Diagnostic Value of Viral Culture and p24 Antigenemia in 181 Infants at Birth.*Article Activity
- Article
THE number of women infected with the human immunodeficiency virus (HIV) is on the increase worldwide, mainly as a result of heterosexual transmission and intravenous drug abuse, and the majority of infected women are of childbearing age. The World Health Organization estimates that by the year 2000, 10 million infants will have been infected, making the prevention and treatment of pediatric HIV disease a major public health challenge.1
Serologic methods, including the enzyme-linked immunosorbent assay (ELISA) and the Western blot assay, are used for the diagnosis of HIV infection in infants over the age of 18 months, but they are not informative at birth because of the presence of maternal antibodies. However, early diagnosis is essential in order to test the effectiveness of early antiviral treatment in asymptomatic children. The study of virologic indexes at birth may help to define prognostic indicators of the rapid and severe form of the disease 2 3 4; it is also important for the parents and the pediatrician, or when adoption is being considered. The sensitivity and specificity of the techniques used to identify infected infants vary greatly during the first three months of life; their validation requires the study of large numbers of infants with adequate follow-up for definitive diagnosis.
We have previously published the protocol and initial results of the French prospective study of infants born to women seropositive for HIV type 1.5 We now present the results of viral culture and p24 antigen assays conducted to identify HIV-1-infected infants at birth and within the first three months of life among the subgroup of infants who underwent tests in the virology laboratory of the Hôpital Necker. Two thirds of the samples from the cohort were sent to this laboratory. Since April 1988, both tests have been applied systematically to all fresh blood samples obtained at birth.
Methods
Patients
Since 1986, more than a thousand infants — all those born to mothers known to be infected with HIV who attended 51 obstetrical and pediatric centers in the Paris, Toulouse, Bordeaux, and Nice areas — have been included in the French prospective study. The only criterion for inclusion in the cohort was documentation of positive maternal HIV serologic status before or at the time of delivery. Clinical and biologic data were collected for each child at birth and at 9, 18, 24, 30, and 36 months.5 Informed consent was obtained from the mothers.
From April 1988 through June 1990, 466 infants were enrolled in the cohort. Twenty infants born to mothers infected with HIV-2, five sets of twins, and six breast-fed infants were considered ineligible for this study. Of 430 singletons born to HIV-1-infected mothers at 43 centers in the Paris area, 291 were enrolled in the 32 such centers that sent blood samples to the virology laboratory of the Hôpital Necker. Cocultures could not be performed for 97 infants, for the following reasons: no samples were received (14 infants); only cord blood was available, the sample was clotted, or the amount of blood was inadequate (56 infants); or there was previous bacterial contamination of the commercial culture medium at the source (27 infants). Cocultures of peripheral-blood lymphocytes for the isolation of HIV-1 were performed for the remaining 194 infants. All these samples were also assayed for the presence of HIV p24 antigen at birth. Seventy-one infants underwent further viral-culture tests before the age of three months, and 86 were retested for p24 antigen. Blood samples were also obtained within 15 days of delivery from 178 of the 194 mothers. The cutoff date for the analysis was chosen as December 1, 1991, so that all the infants would be at least 18 months of age.
HIV Testing
Samples containing 3 to 5 ml of peripheral blood were collected from both the mothers and the infants in heparin-treated tubes within 15 days of birth and were analyzed within six hours. All the results were sent to the attending physicians within six weeks.
Serologic Tests tor HIV-1
The samples were tested for anti—HIV-1 antibodies with two commercial ELISA kits (Elavia-1, available from Diagnostics Pasteur [Marnes-la-Coquette, France] and Abbott Laboratories [Chicago], or Diagnostics Pasteur and Behring [Marburg, Germany]). Confirmatory Western blot assays (Diagnostics Pasteur) were considered positive when antibodies against gp160 and at least two viral core proteins were detected.
HIV-1 Antigen
The specimens from the mother and the infant were systematically assayed for HIV antigen with a sandwich immunoassay (polyclonal antibody, Abbott). Tests were considered positive if the optical density was higher than the cutoff value, calculated according to the manufacturer's instructions, and if the result was confirmed by means of a neutralization test.
Viral Culture
Peripheral-blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation (Milieu de Séparation des Lymphocytes; density, 1.077; Eurobio, Les Ulis, France). Five million PBMCs were stimulated with 5 μg of phytohemagglutinin P (PHA-P; Difco Laboratories, Detroit) per milliliter of solution, were added to 5 million PHA-P—stimulated PBMCs from HIV-seronegative subjects, and were cocultured in upright 25-cm2 flasks (Corning Glass Works, Corning, N.Y.) in 8 ml of RPMI 1640 (Flow Laboratories, Helsinki, Finland, or Gibco, Paisley, Scotland) supplemented with 10 percent fetal-calf serum (Flow), 10 percent (vol/ vol) purified interleukin-2 (Biotest, Frankfurt, Germany), 2 μg of Polybren per milliliter of solution (Sigma, St. Louis), 2 mmol of L-glutamine per liter (Gibco), 100 IU of penicillin per milliliter (Gibco), and 100 μg of streptomycin per milliliter (Gibco). The culture medium was passed through a 0.22-μm filter (Schleicher and Schuell, Dassel, Germany) before use to avoid contamination and was tested for sterility. Fresh PBMCs from healthy donors were prepared twice a week and were used within four days. Half the culture supernatant was removed and replaced with fresh medium twice a week, and the medium was stored at -70°C until use. Two million normal PHA-activated PBMCs were added once a week. Cultures were maintained for six weeks; any cytopathic changes or formation of syncytia was noted each time the medium was changed. Viral replication was monitored by measuring the production of HIV p24 antigen in the culture supernatants with an immunocapture assay twice a week during the first two weeks of culture, then weekly until day 45. On days 3, 7, and 10, 2 ml of culture supernatant was ultracentrifuged at 350,000×g for 10 minutes (TL100, Beckman, Palo Alto, Calif.). The pellet was gently recovered, homogenized in 200 μl of the supernatant, and used in the antigen assay. Thus, there was a 10-fold increase in the concentration of viral particles, and both particle-associated and soluble HIV proteins were assayed. Ultracentrifugation therefore lowered the threshold of antigen detection: supernatants that were weakly antigen-positive after ultracentrifugation (i.e., 1.0 to 2.5 times the cutoff value) were ordinarily negative before it. A coculture was considered positive for HIV-1 when antigen levels exceeded the cutoff value in two consecutive supernatants, one of which was also positive in the neutralization test.
Serologic and Clinical Evaluation of the Infants
An infant was considered to be infected if anti—HIV-1 antibodies (as detected by ELISA and confirmed by Western blotting) persisted beyond 18 months of age or if the infant died of the acquired immunodeficiency syndrome (AIDS). An infant was considered uninfected if HIV-1—specific antibodies disappeared.
The infants infected with HIV-1 were divided into groups according to the classification for children established by the Centers for Disease Control (CDC) on the basis of all symptoms evident during the first 18 months of life, as follows: Class Pediatric-1 (P-1), no symptoms; Class P-2A, nonspecific, general symptoms; Class P-2B, specific encephalopathy; Class P-2C, lymphoid interstitial pneumonitis; Class P-2, category D-1, opportunistic infection; Class P-2, category D-2, severe or repeated bacterial infections; Class P-2, category D-3, other infections; Class P-2E, cancer; and Class P-2F, other conditions, including thrombocytopenia.6
Statistical Analysis
The gold standard for determining the diagnostic values (specificity, sensitivity, and the positive and negative predictive values) of viral culture and antigen testing was the result of serologic tests at 18 months of age. Quantitative variables were compared by Student's t-test. The chi-square test was used for proportions unless any cell count was less than 5, in which case Fisher's exact test was used. All P values are two-tailed, and the threshold of significance was set at 0.05. Results are expressed as means ±SD. When necessary, the tables of Mainland et al.7 were used in determining 95 percent confidence intervals.
Results
Characteristics of the Study Group
The 194 children who underwent viral-culture testing at birth did not differ significantly from the 97 studied during the same period who did not undergo such testing (Table 1Table 1
Characteristics of Mothers and Infants, According to Whether the Infant Underwent Viral Culture at Birth.*). The mean (±SD) age of the 194 infants was 30.3±7.7 months on December 1, 1991. All the infants were born at least 18 months earlier. The infection status of 13 infants (6.7 percent) was unknown. Two infants had died at one month of age: one from unexplained sudden death and the other from causes apparently unrelated to HIV. A further 11 infants were lost to follow-up because the parents moved away shortly after the birth (7 infants) or declined further tests (4).Of the remaining 181 infants, 141 seroreverted (i.e., they lost their antibodies to HIV-1), whereas 40 were considered to be infected (37 were seropositive by the Western blot assay after the age of 18 months, and 3 died of AIDS), giving a transmission rate of 22 percent (95 percent confidence interval, 16 to 28 percent).
Viral Culture
Nineteen of the 181 cocultures carried out at birth (10.5 percent) were positive. The mean ages of the infants were 3.9 days for those who were positive and 5.0 days for those who were negative (P>0.05). A second specimen was tested before the age of 3 months for 11 of these 19 infants (mean age, 32.3±12.7 days), and all were positive. Sixty of the 162 infants with negative cultures at birth were retested before the age of 3 months, and 11 were positive (mean age, 50.2±23.3 days, vs. 42.4±17.4 days for the infants testing negative; P>0.05).
All the cultures became positive by day 3, 7, or 10 of culture. In four cases, viral production was intense immediately and remained so, whereas in other cases the positivity was confirmed by late viral production between days 15 and 38. Only two of the positive cultures had cytopathic changes at the time of p24 positivity.
HIV p24 Antigen Testing
Of the 181 infants tested for HIV p24 antigen at birth, 7 were positive, and all positive results were confirmed by neutralization testing. A second specimen obtained before the age of three months was positive in all four infants tested; in each case, the titer was higher than at birth (210 vs. 76, 366 vs. 185, 651 vs. 445, and 145 vs. 26 pg per milliliter). Of the 174 infants negative for p24 antigen at birth, 81 were tested again before the age of three months, and 4 were positive.
Diagnostic Value of Viral Culture and Antigen Testing
The diagnostic usefulness of viral culture at birth is shown in Table 2Table 2
Diagnostic Value of Viral Culture and p24 Antigenemia in 181 Infants at Birth.* for the 181 infants. The sensitivity was 48 percent (19 of 40) and the specificity 100 percent (141 of 141), whereas the positive predictive value was 100 percent (19 of 19) and the negative predictive value 87 percent (141 of 162). Among the 162 infants with negative cultures at birth, 60 were tested again after an interval ranging from 15 days to 3 months, and 11 had positive cultures. If we considered all infants not retested by culture to be negative (the hypothesis of the maximal bias), the sensitivity of viral culture before the age of three months increased to 75 percent (30 positive cultures among 40 infected infants), and the specificity remained 100 percent (141 negative cultures among 141 who seroreverted).The diagnostic usefulness of HIV p24 antigen testing at birth is presented in Table 2. The sensitivity and specificity of antigen testing at birth were 18 percent and 100 percent, respectively, whereas the positive and negative predictive values were 100 percent and 81 percent. The sensitivity and specificity of repeated antigen testing after an interval ranging from 15 days to 3 months were 28 percent (11 of 40) and 100 percent (141 of 141), respectively, when the same approach was used.
Viral cultures were always positive at birth when p24 antigen was detected in plasma. The four infants who became antigen-positive within three months of birth had positive cultures either at birth or within three months (two infants each).
The sensitivity of the two methods at birth was similar in children with African mothers and in children with mothers from other backgrounds (viral culture, 45 percent [5 of 11] and 48 percent [14 of 29], respectively; p24 antigen testing, 18 percent [2 of 11] and 17 percent [5 of 29]).
Relation between Maternal HIV Antigenemia and Infant Antigenemia and Viral Culture at Birth
Sixteen of 178 mothers (9 percent) had positive HIV p24 antigen tests at the time of delivery. The proportion of infants with positive viral cultures at birth was significantly higher among mothers who were antigen-positive at delivery than among those who were negative (25 percent vs. 7 percent, P<0.04). The difference was more marked among infants positive for p24 antigen at birth. Nineteen percent of infants with antigen-positive mothers were antigen-positive, as compared with 1 percent of those with antigen-negative mothers (P<0.006).
Relation between Virologic Markers at Birth and Progression of Disease
Among the 40 infected infants (Table 3Table 3
Characteristics of the 40 HIV-Infected Children.*), the proportion in whom a severe form of HIV disease (specific encephalopathy or an opportunistic infection) developed was higher in those with positive cultures at birth than in those with negative cultures (32 percent [6 of 19] vs. 14 percent [3 of 21]), but the difference was not significant (P<0.26).Similarly, the proportion of infants who had a severe form of HIV disease was higher among those who were positive for HIV antigen at birth than among those who were negative (57 percent [4 of 7] vs. 15 percent [5 of 33], P<0.04).
Discussion
For the early diagnosis of HIV infection in infants born to infected mothers, various indirect methods based on monitoring of the infant's immune response have been proposed,8 but their sensitivity has not been thoroughly evaluated in large studies. The sensitivity of detection of IgA HIV antibodies has been shown to be increased by the removal of IgG, but still only 30 percent of 23 positive samples from babies three to five months of age were identified.9 , 10 The production of HIV antibodies by cultured lymphocytes can also identify infected infants,11 but not before the age of two months, because of the presence of maternal antibodies.12
In our opinion, early diagnosis is best based on direct detection of the virus, since the immune response can take some time to reach measurable levels. The detection of provirus in peripheral-blood lymphocytes by means of the polymerase chain reaction (PCR) was first proposed in 198813; in one retrospective study, this technique identified 6 of 11 infected infants at birth and 10 of 14 infected infants in the postneonatal period.14
In a prospective cohort study, we performed viral cultures with blood obtained at birth from the infants of 194 seropositive mothers. All the cultures were performed blindly (infected infants are asymptomatic at birth), and the results were given immediately to the attending physicians. The specificity of the cultures was 100 percent, since all samples were negative in the children who reverted to seronegativity, contrary to previous reports.15
We were able to identify about half the infected infants at birth. The detection rate rose to 75 percent when the results of tests done within three months were taken into account, although only 60 of the 162 infants with negative cultures at birth were retested within three months; the remaining 102 children were considered culture-negative.
We attribute the excellent sensitivity of our viral-culture method to four technical factors. Blood samples were cultured rapidly (within six hours); fresh lymphocytes from healthy donors were used as targets, instead of frozen cells; a highly sensitive ELISA was used (instead of reverse transcriptase assays) to detect HIV antigens in the supernatants16; and the supernatants were subjected to ultracentrifugation before the ELISA. Under these conditions, viral antigens were detectable within the first week of culture in 95 percent of positive cases (data not shown), and the cultures could thus be stopped after 14 days. As a result, the findings could be given to the attending physician after a period comparable to that required for PCR. Given the importance of the diagnosis, and despite the excellent specificity of the technique, we always ask for a second specimen to confirm the result before informing the parents. Although one of the major obstacles to viral culture is the need to ensure safety in the working environment, the technique is suited to large-scale analyses by technicians trained in classic methods of cell culture and in ELISA.
The fact that the sensitivity of viral culture is similar in children of African mothers and in those of non-African mothers shows that unlike PCR, the method is not hindered by variation in viral genomes.17 Indeed, the ELISA used to detect antigen in the culture supernatants is effective for a very wide variety of isolates, including those of African origin. A prospective study comparing PCR with viral culture suggested that the former was slightly more sensitive, because it identified 22 of 27 infected infants, as compared with 15 of 27 with the latter technique.18 For our part, we found that the two techniques had similar sensitivities in a blinded study of samples obtained from 35 infants during the first two months of life.19
In the present study, 18 percent of the infected infants were positive for p24 antigen at birth, contrary to previous reports that neonates are antigen-negative.13 , 14 A recent study suggested the possibility of passive antigen transmission.20 But in our experience this test appeared to reflect the presence of viral replication in the infants, because its specificity was 100 percent, even in children born to mothers with detectable antigen at the time of delivery. However, the results of the p24 antigen assay added nothing once viral culture was performed, since all the infants with detectable antigen were also culture-positive. Given its excellent specificity, technical simplicity, and low cost, p24 antigen assay may prove useful when viral culture and PCR are unfeasible. Moreover, the antigen assay should have greater diagnostic usefulness after the dissociation of immune complexes by acid.
In our experience, the sensitivity of viral culture for neonatal HIV infection (48 percent) is far lower than the 97 percent obtained for adults21 and for 102 infected older children (unpublished data), even among those who are asymptomatic and have a low viral burden. This indicates that the number of infected cells is very low during the neonatal period and that HIV transmission probably occurs late in pregnancy or during delivery. This is supported by two facts: in some cases cultures became positive during the first three months of life, and the concentration of p24 antigen increased during the same period. Early HIV transmission has been reported,22 , 23 but its frequency is not known. The apparent absence of the viral genome from fetal tissue,24 the presence of a normal immune system at birth, the absence of neonatal morbidity, and reports of differential viral transmission in twins25 are arguments in favor of late transmission.
It has been reported that a positive result on PCR or culture at birth is predictive of early and severe HIV disease.14 In our study, severe forms of disease developed in a larger number of children with positive cultures or p24 antigen at birth, but the comparison with children in whom slow forms of disease developed was significant only for those with p24 antigen. This is not surprising, since the sensitivity of the culture was 48 percent and only about 20 percent of the children had early and severe forms of the disease.5
In conclusion, viral culture is a reliable method of identifying a sizable proportion of infected infants at birth. Studies based on quantitative variants of this technique should provide further information on both the most likely moment of vertical transmission and the predictive clinical value of such tests.26
Supported by grants from the Agence Nationale de Recherche contre le SIDA.
*The following persons participated in the HIV Infection in Newborns French Collaborative Study: Drs. C. Allisy, D. Armengaud, J.M. Babinet, P. Balde, F. Ben Fadel, R. Bensadoun, D. Berterotiere, M. Boddaert, Y Bompard, C. Botto, A.M. Boulley, C. Brunner, J.A. Cacault, C. Carlus-Montcomble, C. Cohen, A.M. Colin-Gorski, C. Courpotin, C. Crumiere, M.C. Dallot, M. Dandine, A. De Crepy, M. Debons, M. Debre, M.F. Denavit, A. Devidas, M. Dumondel, C. Francoual, J. Furioli, A. Gantzer, F. Granier, B. Heller, C. Huraux-Rendu, P. Labrune, A. Lacroix, M. Lanza, H. LeJalle, B. Le Lorier, A. Leblanc, F. Lebrun, C. Lejeune, J. Mamou, V. May, F. Mazi, A. Meyer, G. Mouchnino, P. Narcy, G. Noseda, C. Pascal, B. Pautard, M. Robin, M. Ronzier, J.L. Ropert, M.C. Rousset, A. Saillant, G. Scart, H. Seaume, D. Seguy, P. Talon, M. Tardieu, J. Terris, L. Valdes, F. Veber, J. Vedrenne, E. Vilmer, A. Vinas, N. Vincent, and P. Wipff. The study coordinators were C. Rouzioux (Virology), S. Blanche (Clinic), and M.J. Mayaux (Epidemiology).
We are indebted to David Young, Corinne Laurent, Anne Doussin, Patricia Rosinel, Richard Larchée, and the technicians of the Laboratoire de virologic of the Hôpital Necker—Enfants Malades for their excellent technical assistance, and to Marie-Laurence Fourneaux and Rosine Fournier for their assistance in the preparation of the manuscript.
Source Information
From the Laboratory of Microbiology (M.B., A.F., C.R.), the Immunology and Hematology Unit, and INSERM U.132 (S.B., C.G.), Hôpital Necker—Enfants Malades, Paris; INSERM U.292, Hôpital de Bicêtre, Kremlin-Bicêtre (M.-J.M., M.-L.G.-M.); and the HIV Infection in Newborns French Collaborative Study (M.C.A., N.C.-V., G.F., C.F., F.G., E.L., M.V.) — all in France. Address reprint requests to Dr. Rouzioux at the Laboratoire de virologic, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France.
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