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False-negative syphilis screening: The

prozone phenomenon, nonimmune hydrops,
and diagnosis of syphilis during pregnancy
Article in American Journal of Obstetrics and Gynecology October 1990
DOI: 10.1016/0020-7292(91)90336-4 Source: PubMed

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False-negative syphilis screening: The prozone phenomenon,

nonimmune hydrops, and diagnosis of syphilis
during pregnancy
Kathleen Berkowitz, MD, Laxmi Baxi, MD, and Harold E. Fox, MD
New York, New York
The prevalence of congenital syphilis is rapidly rising in several areas of the United States. Efforts to
control the disease depend on the effectiveness of established screening strategies and treatment of
infected pregnant women. False-negative test results hinder these efforts and leave the fetus at risk for
acquiring congenital syphilis. Recently we encountered four cases of false-negative syphilis serologic
results in women who gave birth to infants with congenital syphilis. The false-negative results were caused
by the prozone phenomenon. The prozone phenomenon, seen during primary and secondary syphilis,
occurs because a higher than optimal amount of antibody in the tested sera prevents the flocculation
reaction typifying a positive result in reagin tests. Serum dilution is necessary to make the correct
diagnosis. We recommend that for any pregnant woman with apparently negative syphilis serologic results
in whom fetal compromise of unknown etiology exists, particularly nonimmune hydrops, nontreponemal
testing should be repeated using serum dilutions to prevent a missed diagnosis of syphilis. We further
recommend serum dilution as a routine procedure for all pregnant women in areas of high syphilis
prevalence. (AM J OBSTET GVNECOL 1990;163:975-7.)

Key words: Prozone phenomenon, false-negative syphilis screening, serum dilution,

screening strategies, nonimmune hydrops

A dramatic increase in the prevalence of syphilis has

been noted in several areas of the United States during
the past 2 years. The prevalence of congenital syphilis
has also risen rapidly and in some areas is now considered epidemic. 1 Attempts to control this epidemic hinge
on adequate surveillance, identification, and treatment
of infected women.
The use of nontreponemal serologic tests is widely
accepted as an effective screening strategy for the detection of syphilis during pregnancy. However, in some
clinical situations difficulty in diagnosis results in misidentification of the true disease state despite the use
of highly accurate tests. False-positive results have been
reported in many conditions including pregnancy.2-5 Serial testing, dark-field examination, or treponemal tests
clarify the diagnosis in these situations. False-negative
results may occur when the patient is tested long after
treatment, during late-latent syphilis, or as a result of
the prozone phenomenon. A false-negative error presents a serious problem because an infected woman
From the Department of Obstetrics and Gynecology, Sloane Hospital
for Women, Columbia Presbyterian Medical Center.
Presented at the Tenth Annual Meeting of the Society of Perinatal
Obstetricians, Houston, Texas, January 23-27, 1990.
Reprint requests: Kathleen M. Berkowitz, MD, Department of Obstetrics and Gynecology, Culumbia Presbyterian Medical Center, 622
W. 168th St., New York, NY 10032.
616122357

would then fail to receive treatment, leaving her fetus

vulnerable to the disastrous consequences of congenital
syphilis.
The prozone phenomenon occurs when an excess of
antibody in the sera being tested prevents the formation
of the antibody-antigen lattice network needed to visualize a positive flocculation reaction. 6 Antibody may
remain undetected if the antigen is too dilute or too
concentrated. The zone of concentrations in which precipitation or flocculation can occur is called the "optimal
zone." When a sample shows a negative reaction because of antibody-antigen concentrations outside the
optimal zone, a "zonal reaction" or prozone phenomenon has occurred. 7 The term "sero-negative syphilis"
was coined in 1950 to describe the effect. 8 The prozone
phenomenon can affect any agglutination reaction and
its first description in 19079 predates even the Wassermann complement fixation test. The prozone phenomenon can cause a false-negative result when Venereal
Disease Research Laboratories or rapid plasma reagin
tests are used and occurs in approximately 2% of cases
of primary or secondary syphilis. 1O Serum dilution reestablishes the proper concentrations of antibody and
antigen that allow lattice formation.
Four women with false-negative syphilis serologic results (Becton-Dickinson rapid plasma reagin card test,
Rutherford, N.J.) gave birth to infants with congenital
975

976

Berkowitz, Baxi, and Fox

syphilis at the Sloane Hospital for Women in 1989. The

false-negative results were caused by the prozone phenomenon. This article details their clinical presentations and the management difficulties encountered as
a result of the prozone phenomenon.

Case reports
Case I. A 26-year-old woman (gravida 8, para 3-04-2) was seen at 32 weeks' gestation with a complaint
of decreased fetal movement. Rapid plasma reagin testing at 16 weeks of gestation and in previous years had
been reported as negative. There was no maternal clinical evidence of syphilis. TORCH titers and a rapid
plasma reagin test result were reported as negative on
admission. Ultrasonography revealed bilateral hydrocele, polyhydramnios, and possible hydrops. During
cordocentesis for karyotyping and acid-base determination, bradycardia and prolonged umbilical cord
bleeding occurred that necessitated emergency cesarean section. A live-born male infant weighing 2525 gm
with Apgar scores of 0 and 3 at 1 and 5 minutes was
delivered. The infant had significant metabolic acidosis,
thrombocytopenia, and physical signs of congenital
syphilis. There was no cord hematoma. A second
maternal serologic sample (Becton-Dickinson rapid
plasma reagin card test) yielded an initial negative result and a positive titer of 1 : 64 on serum dilution; the
treponemal test result was positive.
Case 2. A 21-year-old woman (gravida 3, para 1-01-1) at 32 weeks' gestation who had received no prenatal
care was seen with malaise and decreased fetal movement. There was no clinical evidence of syphilis and
she reported past negative serologic results. Fetal heart
rate monitoring revealed a minimally reactive tracing.
Amniotic fluid was increased and the placenta appeared edematous on ultrasonogram. Rapid plasma reagin and TORCH titers were reported as negative on
admission. The original serum sample was diluted to
evaluate a possible prozone phenomenon because of
strong clinical suspicion of intrauterine infection. A cesarean section was performed for management of fetal
distress. A male infant weighing 1460 gm with Apgar
socres of 5 and 8 at 1 and 5 minutes was delivered. The
infant had clinical and serologic evidence of congenital
syphilis. Long-term follow-up of this infant is not available. Serum dilution titer was 1 : 256 with positive treponemal testing.
Case 3. A 36-year-old woman (gravida 6, para 4-01-0) was seen at term in active labor. She had no clinical
or historical evidence of syphilis infection. A perineal
lesion seen 1 week earlier was positive after culture for
herpes simplex virus and a rapid plasma reagin test on
admission was reported as negative. Fetal distress was
seen on admission and a cesarean section delivery produced a male infant with Apgar scores of 1 and 1 at 1
and 5 minutes. The infant had petechiae, hepatosplenomegaly, thrombocytopenia, and died 7 hours
post partum. Pathologic studies showed a pale and
bulky placenta. Serum dilution yielded a postive titer
of 1: 128 with positive treponemal testing.

September 1990
Am ] Obstet Gyneco1

Case 4. A 30-year old woman (gravida 4, para 1-02-1) at 27 weeks' gestation had contractions and decreased fetal movement. She had received no antenatal
care and had a history of adequately treated primary
syphilis in 1986. The rapid plasma reagin test for syphilis on admission was negative despite physical findings
including condyloma lata. Ultrasonography confirmed
a 27 -week intrauterine death with hydrops and placentomegaly. The patient was spontaneously delivered of
a severely macerated, hydropic fetus weighing 2070
gm. Petechia, splenomegaly, and funisitis ll were noted
at autopsy and the placenta was bulky and pale. Serum
dilution yielded a rapid plasma reagin titer of 1: 256
with positive treponemal testing.

Comment
The laboratory diagnosis of syphilis can be made with
darkfield microscopy or serologic testing. The mobile,
corkscrew-shaped organisms of Treponema pallidum can
be identified microscopically by examination of fresh
exudate with a dark-field condenser. Serologic tests
are divided into treponemal and non treponema! tests.
Treponemal tests detect antibodies against T. pallidum
by incubation of the specimen with labeled antiT. pallidum globulin (fluorescent treponemal antibodyabsorption test) or sensitized sheep erythrocytes (microhemagglutinin test). The first treponemal test developed in 1949 was the T. pallidum immobilization test.
It has been largely replaced by the more sensitive, less
expensive tests mentioned earlier. The nontreponemal
tests, such as the VDRL or rapid plasma reagin card
test, use purified cardiolipin-lecithin-cholesterol antigen. The VDRL test requires heated serum and a positive result is determined microscopically by visualizing
flocculation. The rapid plasma reagin card test and the
automated reagin test use unheated serum and stabilized cardiolipin antigen suspended in charcoal. The
results are read macroscopically as a result of agglutination. This approach has the advantage of being simpler to perform and standardize. When evaluating the
efficacy of treatment by measurement of serum titers,
the same nontreponemal test should always be used
because there may be a marked variation in titers between tests. The nontreponemal tests generally are
more sensitive in the diagnosis of primary syphilis, but
in secondary syphilis there is no difference in sensitivity
between nontreponemal and treponemal tests.12 Treponema! tests are more sensitive in the diagnosis of tertiary syphilis.
The prozone phenomenon occurs when the amount
of antibody in a particualr serum sample is too high to
allow formation of the antibody-antigen complex necessary to visualize flocculation or agglutination. Syphilitic infection may be hidden in those patients whose
serologic results are rendered negative by the prozone
phenomenon. This is a particulary worrisome problem
in asymptomatic pregnant women. Because the true

False-negative syphilis screening

Volume 163
Number 3

cause of fetal compromise is masked, the management

of pregnancy and labor cound potentially be misguided. Diagnosis of congenital syphilis with its attendant risk of thrombocytopenia and anemia affect management of the timing and route of delivery. The last
case illustrates that routine serologic study may be misleading in the diagnosis of inadequately treated or reacquired disease.
The predictive value and cost benefit of screening
pregnant women for syphilis depends on the prevalence of the disease in the population being tested. Because the cost of treating infants with congenital syphilis
is so high, screening and treatment of almost all populations of infected women is cost beneficial." The
prevalence of syphilis in the population of women who
were delivered at the Sloane Hospital for Women in
1988 was 2.7% (140 cases in 5280 deliveries). Of the
infants born to these women, at least seven (1.3 per
1000 deliveries) had symptomatic congenital syphilis.
Approximately two thirds were asymptomatic but had
serologic test results compatible with the diagnosis of
congenital syphilis and required intravenous antibiotic
therapy.
We recommend that serum dilution be performed to
evaluate the possibility of a false-negative result caused
by the prozone phenomenon for any woman with negative serologic results in whom intrauterine infection is
suspected. Serum dilution should also be included in
the workup of nonimmune hydrops. When clinically
indicated, ultrasonography is helpful in discovering evidence of intrauterine infection such as placentomegaly,
polyhydramnios, or intrauterine growth retardation.
We further recommend that in populations with a high
prevalence of syphilis every woman should have rapid
plasma reagin testing with serum dilution on presen-

tation for antepartum care and again

trimester.

III

977

the third

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