712
Defining the Time of Fetal or Perinatal Acquisition of Human Immunodeficiency
Virus Type 1 Infection on the Basis of Age at First Positive Culture
Leslie A. Kalish, Jane Pitt, Judy Lew,
Sheldon Landesman, Clemente Diaz, Ronald Hershow,
F. Blaine Hollinger, Marcello Pagano, Vincent Smeriglio,
and Jack Moye for the Women and Infants Transmission
Study (WITS)
New England Research Institutes, Watertown, and Harvard School of
Public Health, Boston, Massachusetts; Columbia University, College of
Physicians and Surgeons, New York, and State University of New York
Health Science Center, Brooklyn, New York; National Institute of
Allergy and Infectious Diseases, National Institute on Drug Abuse, and
National Institute of Child Health and Human Development, National
Institutes ofHealth, Bethesda, Maryland; University of Puerto Rico, San
Juan, Puerto Rico; University of Illinois at Chicago, Illinois; Baylor
College of Medicine, Houston, Texas
It has been suggested that a positive diagnostic test for human immunodeficiency virus type 1
(HIV-1) during the first 48 h of life is indicative of intrauterine transmission, whereas negative tests
during the first week with positive tests later indicate intrapartum transmission. On the basis of
data from all 140 infected infants in the Women and Infants Transmission Study (WITS), the
probability was estimated that an HIV-1 culture would be positive for the first time at each day of
life if cultures were performed daily. The estimated probabilities (±SE) by days 0, 2, 4, 7, 9, 16,
and 30 of life are 27.4% (±6.4%), 27.4% (±13.0%), 45.3% (±20.5%), 45.3% (±22.5%), 65.3%
(±20.0%), 88.4% (±7.8%), and 89.3% (±7.0%), respectively. The initial 27% probability is consistent
with the hypothesis that transmission usually occurs during the intrapartum period. However, the
distribution of age at first positive culture does not separate clearly into two distinct intervals. More
definitive methods for determining the timing of transmission are needed.
The timing of perinatal human immunodeficiency virus type
1 (HIV-1) transmission is a topic of interest in the field of
pediatric HIV research. Most estimates of the proportion of
infections that occur during the intrauterine period range from
~25% to ~40% [1-4], with one estimate as high as 60% [5].
Bryson et a1. [6] have proposed a working definition that
classifies vertical transmission of HIV-1 as occurring in the
intrauterine or intrapartum period. Briefly, an infected infant
whose peripheral blood mononuclear cells (PBMC) are positive
Received 21 May 1996; revised 30 September 1996.
Presented: XI International Conference on AIDS, Vancouver, Canada, 711 July 1996.
Informed consent was obtained from the parents or guardians of subjects in
this study. Human experimentation guidelines of the US Department of Health
and Human Services and of the authors' institutions were followed.
Principal investigators, study coordinators, program officers, and funding
(NIH) include Clemente Diaz and Edna Pacheco-Acosta (University of Puerto
Rico, San Juan; AI-34858); Ruth Tuomala, Ellen Cooper, and Donna Mesthene
(BostonIWorcester Site, Boston; AI-34856); Jane Pitt and Alice Higgins (Columbia Presbyterian Hospital, New York; AI-34842); Sheldon Landesman,
Hermann Mendez, and Gail Moroso (State University of New York, Brooklyn;
HD-829l3, HD-257 14); Kenneth Rich and Delmyra Turpin (University of
Illinois at Chicago; AI-34841); William Shearer, Celine Hanson, and Norma
Cooper (Baylor College of Medicine, Houston; AI-34840); Mary Glenn Fowler
(National Institute of Allergy and Infectious Diseases); Anne Willoughby (National Institute of Child Health and Human Development); Vincent Smeriglio
(National Institute on Drug Abuse); and Sonja McKinlay and Kathy Sherrieb
(New England Research Institutes, Watertown, MA; AI-35161).
Reprints or correspondence: Dr. Leslie A. Kalish, New England Research
Institutes, 9 Galen St., Watertown, MA 02172; e-mail: [email protected].
The Journal of Infectious Diseases
1997; 175:712-5
© 1997 by The Universityof Chicago. All rights reserved.
0022-1899197/7503 -0033$0 1.00
by culture or by the polymerase chain reaction (PCR) within
48 h of birth is classified as having intrauterine transmission,
whereas transmission is classified as intrapartum if diagnostic
studies are negative during the first week of life. These criteria
are based on the assumption that a time interval is required
between the actual transmission event and the ability to detect
the infection. This lag period presumably is defined by the time
required for whatever number of cycles of cellular infection
and viral replication is needed to achieve circulating levels of
cell-associated virus sufficient to be detected by tissue culture
or proviral PCR methods. With intrauterine transmission, the
lag time presumably has elapsed by the time of birth.
Statistical analysis of relevant data to address this issue is
complicated by "interval censoring." For example, for an infant with a negative diagnostic test at age 2 days and a positive
test at age 30 days, the age at which the infant would first test
positive if monitored daily is only known to be in the interval
from 3 to 30 days.
Estimates of the probabilities of a positive diagnostic test at
various ages provide a tool for evaluating the proposed definition and for refining our understanding of HIV-1 transmission.
Dunn et a1. [3] used nonparametric methods for interval-censored data and estimated the sensitivity of proviral DNA PCR
for detecting HIV-l during the neonatal period in 271 infected
infants pooled from 12 sources. We performed a similar analysis using HIV-1 cultures from 140 infected infants enrolled in
the Women and Infants Transmission Study (WITS), an ongoing cohort study of HIV -1- infected pregnant women and their
children [7]. The purposes were to estimate the probability, at a
given age, that an infected infant would first produce a positive
Concise Communications
JID 1997; 175 (March)
Ql
100
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713
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result would be positive for the first time.
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60
30
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7
14
21
28
35
42
Age (days)
Figure 1. Cumulative probability of 1st positive HIV-1 culture.
Vertical bars represent 90% confidence intervals.
culture if cultures were obtained frequently and to assess the
roles of intrapartum and intrauterine transmission.
Methods
Enrollment in WITS began in December 1989. This analysis
includes all 140 WITS infants who were classified as HIV-1infected on the basis of data collected through 30 November 1995.
None of these infants were breast-fed. Peripheral blood was collected for HIV-1 culture at or shortly after birth and at nominal
ages 1, 2, 4, and 6 months. Permissible windows around nominal
ages were 7 days for the birth visit, ::±::2 weeks for the 1- and 2month visits, and ::±::4 weeks for subsequent visits. Since April
1994, the protocol called for two' 'visit 1" cultures, the first during
days 0-1 and the second during days 6-10 of life. After 6 months
of age, infants with positive or indeterminate infection status were
to have cultures repeated every 6 months (::±::4 weeks).
Samples were collected in heparinized tubes and transported at
ambient temperature to a local virology laboratory, where PBMC
were separated within 18 h of collection. Cultures were performed
qualitatively until October 1991 and quantitatively thereafter [8].
HIV-1 infection was determined by the presence of ~2 positive
PBMC cultures. For purposes of classifying overall infection status, a quantitative culture was considered positive if ~2 wells
were positive, at least 1 of which was in the first 4 wells (i.e., first
2 dilutions). For an infant who met these criteria for infection, a
culture with a single positive well was also considered positive,
if the positive well was among the first 4 wells (i.e., first 2 dilutions). The AIDS Clinical Trials Group (ACTG) consensus PBMC
culture protocols were used [8], and all laboratories participated
in the ACTG virology quality assurance program [9].
The age at which a culture would first be positive if cultures were
performed every day oflife cannot be known exactly. However, the
age can be bounded by the interval ending at the earliest positive
culture and beginning at the latest negative prior culture (or at
birth if there are none). Nonparametric methods for estimating the
distribution oftime-to-event data from interval-censored data [10,
At each study visit through 6 months, at least 100 infants
had a culture result. Although the protocol called for two visit1 cultures since 1 April 1994, only 16 infants in this analysis
were born after this time. Thirteen had a second visit-1 culture,
a median of 6 days after birth. In 12 of 13 cases, the 2 culture
results were concordant (6 double positives, 6 double negatives). One was negative on day 0 and positive on day 9.
Therefore, the close sampling points were not very helpful in
narrowing the change-over time from negative to first positive
result in these data.
The estimated cumulative probability of a first positive culture is shown in figure 1, along with 90% confidence intervals.
The estimated probabilities (::!::SE) of a first positive culture by
days 0, 2, 4,7,9,16, and 30 days of life are 27.4% (±6.4%),
27.4% (::!::13.0%), 45.3% (::!::20.5%), 45.3% (::!::22.5%), 65.3%
(±20.0%), 88.4% (::±::7.8%), and 89.3% (::±::7.0%), respectively.
Table 1 shows the number of cultures done and the numbers
and percentages positive, by age. Thirty-eight percent (15/40)
of cultures obtained on the day of birth were positive. This
prevalence decreased to 17% (4/23), 15% (2/13), and 0% (0/
8) 1, 2, and 3 days later, respectively. There are no repeated
observations for an individual infant during these early time
points, so these are purely cross-sectional estimates. The decreasing trend may well represent sampling variability. During
days 4-9 of life, 57% (20/35) of cultures were positive and
the prevalence was 89%-93% at months 1-6 of age.
Table 1. Cross-sectional prevalence of positive cultures in HIV-1infected infants by age.
Age
No. of cultures
No. positive (%)
40
23
13
8
15 (38)
4 (17)
2 (15)
0(0)
4 (67)
4 (80)
7 (50)
2 (40)
1 (100)
2 (50)
89 (89)
114 (93)
100 (88)
104 (93)
Days
o
1
2
3
4
5
6
5
6
14
5
7
8
1
4
100
123
114
112
9
1m ± 2w
2m ± 2w
4m±4w
6m ± 4w
NOTE.
m, months; w, weeks.
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Concise Communications
Discussion
If cultures obtained before a certain age in an infant who
was infected intrapartum were never positive, then we would
expect to see a constant probability in figure 1 up to that age,
followed by an increase. If all cases of intrapartum transmission
had the same delay to when a culture would first be positive,
then this increase would be very steep. The age at which the
sharp rise in positivity occurs would permit investigators to
classify the timing of transmission as intrauterine or intrapartum, on the basis of earlier culture results. However, the analysis suggests that the distribution of age at first positive culture
does not separate into 2 clearly distinct age intervals. The
estimated 27% probability of a positive culture during the first
few days of life is consistent with most prior estimates. Large
SEs during the latter part of the first week make it difficult to
establish a sharp cutoff point between 2 distinct populations
(intrauterine and intrapartum transmission), although cultures
from 85%-90% of infected infants should be positive at least
once during the first 2 weeks of life if these infants are sampled
repeatedly. More frequent sampling time points for each infant
would have provided a more precise estimate of the curve in
figure 1 and may have allowed for definition of a sharper cutoff
point.
The proviral DNA peR data of Dunn et al. [3] suggest that
those who are negative early in life become positive during
the second week of life. They estimated that 38% of infected
infants test positive on the day of or the day after birth. Sensitivity remained fairly constant during the first week, rising to 45%
on days 4-7 of life, and then increased to 93% by 14 days.
Our estimates are consistent with these results. However, confidence intervals during the latter part of the first week in our
data are wide, so our data are also consistent with an increase
during the first week.
On the basis of a parametric Markov model, Rouzioux et al.
[4] estimated that the percentages positive by culture or PCR
on days 0, 2, 7, and 10 are 17%,25%,42%, and 50%, respectively. They estimated that 35% of infants are infected in utero.
There is no sharp increase over time in these results, but their
assumed model does not allow the curve to have this shape.
The probability at a particular age shown in figure 1 is not
an estimate of the sensitivity of culture at that age for detecting
infection status but represents the probability of observing at
least 1 positive culture up to that age if cultures were done
frequently. Thus, the estimates eventually reach 100%, although culture is only 88%-93% sensitive at 1-6 months of
age (table 1). If a positive culture in a particular infant was
never followed by a negative culture, then the probabilities in
figure 1 would be estimating sensitivity. Dunn et al. [3] noted
no positive followed by negative results in their DNA PCR
data. The specificity of culture for determining overall infection
status has been demonstrated to be very high [12].
If culture on the day of birth was a perfectly accurate method
for distinguishing intrauterine from intrapartum infection, then
JID 1997; 175 (March)
the observed proportion positive at birth would be an unbiased
estimate of the intrauterine infection rate. The possibility of a
positive culture at birth in an infant who was infected intrapartum
seems remote (i.e., specificity is high). However, even months
after infection is established, the sensitivity of a single culture
is only ~90% (table 1). In addition, a transmission event shortly
before delivery (in utero) may not have sufficient time for infection to be established and to produce a positive culture at birth.
Therefore, < 100% of intrauterine-infected infants can be expected to test positive at birth, so the observed proportion positive at birth will underestimate the true proportion experiencing
intrauterine transmission. For example, if 80% of intrauterineinfected infants test positive at birth, then an unbiased estimate
of the proportion infected would be the observed proportion
positive inflated by a factor of 1/0.80. Unfortunately, there is
currently no way to determine the inflation factor unless one
makes additional modeling assumptions [4].
One conservative approach would be to assume that only
patients with cultures positive on the day of birth represent
infections acquired in utero. Definitive classification of all individual cases on the basis of a single culture on the day of
birth remains problematic, however, because the sensitivity of
current methods is < 100%, as described above. Negative results on repeated sampling at birth and during the first days of
life would provide greater specificity for detecting intrauterine
transmission.
The statistical method does not yield a classification of intrauterine or intrapartum transmission for each individual, only
an estimate of the population distribution of times at which a
culture would first be positive if monitored "continuously."
In practice, it is often difficult to apply the criteria of Bryson
et al. [6], due to the temporal spacing of diagnostic studies of
any particular infant.
In addition to characteristics inherent in the detection assays
and their application, factors intrinsic to either the host or virus
may influence the time period between a transmission event
and culture or DNA PCR positivity. These may include the
inoculum size, genotypic features of the infecting virus strain,
host genetic and other determinants of target cell susceptibility
and immune response, and the recommended use of antiretroviral chemoprophylaxis. Recent studies of HIV-infected adults
[13] suggest that the kinetics of viral replication are exceptionally dynamic. A better understanding of these parameters in
primary infection of the fetus and newborn may provide further
insight.
The timing of transmission may have prognostic implications with respect to clinical disease progression [14]. Currently, the time at which HIV -1 infection first becomes detectable is being used as a surrogate for timing of
transmission. This surrogacy may be inadequate. More rigorous efforts are needed to define the time at which HIV-I
infection first becomes detectable and the time at which HIV1 transmission occurs. Whether these are separate questions
remains to be determined.
JID 1997; 175 (March)
Concise Communications
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