Antiviral
Chemotherapy
and Neonatal
Herpes
Simplex
Virus
Infection:
A Pilot
Study-Experience
With
Adenine
Arabinoside
(ARAA)
Lawrence T. Ch’ien, M.D., Richard J. Whitley, M.D., Andre J. Nahmias, M.D., Edward
B. Lewin, M.D., Calvin C. Linnemann, Jr., M.D., Lawrence D. Frenkel, M.D., Joseph A. Bellanti, M.D., Robert A. Buchanan, M.D., and Charles A. Alford, Jr., M.D.
From the Department of Pediatrics, University of Alabama, Birmingham; Emory University, Atlanta; the
University of Cincinnati, Cincinnati, Ohio; Georgetown University, Washington, D.C.; the University of’ Rochester, Rochester, New York; and Parke, Davis and Co., Ann Arbor, Michigan
ABSTRACT. Among 13 neonates with herpes simplex virus
(HSV) infection, eight had disseminated disease, one
local-ized CNS disease, and in four the infection was confined to the skin and eyes. Ara-A, a purine nucleoside with anti-viral activity against DNA-viruses, was given (10 to 20 mg/kg/ day) by a continuous 12-hour intravenous drip for 10 to 15 days. In all, ara-A administration was begun within three to eight days after the appearance of skin vesicles which represented the hallmark of the disease. Both diagnosis and ara-A treatment were much delayed in one infant without skin vesicles and four infants whose skin vesicles appeared late, long after the occurrence of CNS damage. In this group of infants with disseminated disease, four died and one infant was left with severe neurological deficits.
Eight infants (four with disseminated and four with localized skin disease) with skin vesicles as the earliest sign of infection received ara-A early, within three days after the onset of neurologic signs. All survived with no neurologic deficit at 6 months to 1 year of age. There was no apparent toxicity of ara-A to the bone marrow, liver, or kidney.
Pediatrics, 55:678, 1975, ANTIVIRAL CHEMOTHERAPY,
NEO-NATAL HERPES, HERPES SIMPLEX VIRUS, ADENINE
ARABINO-SIDE.
Although a few cases of neonatal herpes may be acquired
in utero,38
the large majority of cases are a result of intrapartum infection. Characteristi-cally, damage to the brain or other vital organs is absent at the time of birth, unlike the chronic intrauterine infections associated with toxoplas-mosis, rubella, or cytomegaloviruses. Therefore, the disease is potentially amenable to therapy if instituted early.During the past few decades, the massive screening of antiviral substances has resulted in only a few clinically accepted compounds. Two antiviral agents-idoxuridine (IDU) and cytosine arabinoside (ara-C)-have been employed in cases
of
severe herpes simplex virus infections, includ-ing neonatal herpes.97 The use of IDU and ara-C has been limited to infants with severe disease because of their acute toxicity to the boneNeonatal herpes simplex virus (HVS) infection. which is associated with high rates of morbidity and mortality, is usually contracted from the infected maternal birth canal.1 Since most cases of maternal herpetic infection are not clinically recognized, performing a cesarean section in attempt to prevent the infant from being exposed to the virus is often not possible.2
(Received July 16; revision accepted for publication September 12, 1974.)
The studies performed at the University of Alabama were supported by grants NICHD HD01687 and Ca 13148 from the National Cancer Institute and by Parke Davis and Co.
The Emory studies were supported by the National
Founda-tion.
TABLE I
ExcRETIoN OF HSV Fiio,t ThE THROAT DURING ARA-A ADMINISmATI0N
Patient VinisTx
Vin&s Titer Thma t Swab Material#{176}
Pretn’,atment Day5 Day7 Day 18
2 HSV-2 3.5 2.5 Negative Not done
3 HSV-2 3.5 3.5 2.0 1.0
5 HSV-1 6.5 2.5 Negative Not done
#{176}Log10TCID/0.2 ml.
marrow and the host immunological system. A third agent, adenine arabinoside (ara-A) which is a purine nucleoside also found effective
in vitro
and in experimental animals against herpesviruses including HSV,’822 has not been previously reported in the treatment of neonatal herpes.
When ara-A is given parenterally, it enters the cerebrospinal fluid readily.23 Within its antiviral dose range in man, it does not appear to suppress the bone marrow or the host immune system.24 Its first-step metabolite, ara-hypoxanthine, retains antiviral activity, though less than the parent compound. Some derivative tends to persist intra-cellularly and may provide a prolonged antiviral effect.2425 In this report, we are presenting the results of ara-A treatment of 13 newborns with
HSV infection.
MATERIAL AND METHODS
Patients
Thirteen newborn infants with virologically documented HSV infection were admitted to the study at various stages of their illness. They can be
classified into three groups on the basis of clinical findings and time of administration of ara-A: group I (cases 1 to 4) and group II (cases 5 to 9) had disseminated infection or localized CNS disease. The disease was considered disseminated when skin, CNS, and one or more visceral organ systems were involved. The disease was defined as
“localized CNS” when only skin and CNS involvement were evident. Group I received ara-A earlier than group II in the course of the disease. Group III (cases 10 to 13) had localized infection involving the skin and/or eyes only.
Laboratory Methods
Bacterial and fungal cultures were routinely performed on each cerebrospinal fluid specimen. Multiple specimens for viral culture were obtained as soon as there was clinically suspected
herpetic infection. In most cases, the following specimens were cultured for virus: the throat
swab material, urine, feces, conjunctivae, and
peripheral blood buffy coat cells.
For isolation of HSV, primary rabbit kidney
cells or diploid human fibroblasts were employed. Inoculated cell cultures were observed for
cyto-pathic effect for at least one week before they
were discarded as negative. Serotyping of HSV
employing immunofluorescent technique was performed according to procedures previously described.26
Ara-A Administration
Ara-A, supplied by Parke, Davis and Company,
was administered intravenously at a steady rate
over 12 hours in dosages ranging from 10 to 20
mg/kg/day for 5 to 15 days. During drug
admin-istration, toxicological monitoring including daily clinical evaluations and laboratory assessment of
peripheral blood elements and hepatorenal
func-lion at five-day intervals.
RESULTS
Patient Population
The mothers of affected infants were
predomi-nantly young with an age range of 15 to 25 years
(mean age, 18 years), black (eight black and five
white); and primiparous (8 of 12 or 66%). The
mean birthweight of the 13 infants was 2,778 gm.
Four infants weighed less than 2,500 gm at birth.
Nine were male infants and four were females.
Maternal
Genital Infection
Nine of the 13 mothers did not have any history
of genital herpes or signs of herpetic infection at
delivery. A history consistent with maternal
genital herpetic infection during pregnancy was
elicited from only two mothers.
The mother of patient 2 had pelvic pain and
NEONATAL HSV INFECTION
I 5 10 15 20 25 30 35
[0000000000000(
40 45
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[ooooooooooo
122ooo000000002.2J EJ LJ
d 2
I-2
w 2
3
4
5
6
7
8
9
10
II
2
‘3
RECOVERED
RECOVERED
RECOVERED
RECOVERED
10000001
- ANA-A THERAPY
SEIZURES aNEURO SIGNS L______JNEURO. SIGNS
tooooooc SKIN VESIcLES
tuu” ANTIBIOTIC THERAPY
L I
ii:
DISSEMINATED
DIED
DIED
DIED
Lz*zxxixxxxi*xx ***;
I
IZ*&ZI*XX**I*****R**
DIED
5’
“>““‘“ MICROCEPHALY
[#{224}ioooooooI
::i:
RECOVEREDoooooooI LOCALIZED RECOVERED
(ALL HSV-l)
[ooooooooooooo( RECOVERED
Ei:i::i:i:oooo (DendriticRECOVEREDCorneal Ulcer)
IllIllIllI II II I__I_ II II II II
I 5 10 15 20 25 30 35 40 45
AGE (DAYS)
Ftc. 1. Temporal relationships between the onset and duration of skin vesicles, nonspecific neurologic signs, seizures, antibiotic
therapy, and ara-A therapy.
labor. The father also suffered from genital herpes. Neither sought medical attention.
The mother of patient 6 was followed for genital herpes during pregnancy. HSV was cultured from vaginal vesicles at 33 weeks; culture of the amniotic fluid was negative for HSV at 35 weeks. Spontaneous rupture of membranes occurred at 36 weeks and the infant was delivered 15 hours later by cesarean section. Fluid obtained by amniocentesis immediately before the cesarean section grew HSV and group
B streptococci.
In two other mothers, the maternal genital
infection was clinically inapparent. Papanicolaou smear from the cervix of one mother (patient 4’s mother) showed inclusion-bearing cells character-istic of HSV one week before delivery. The infection was silent and, unfortunately, the cyto-logical report did not reach the physician until the mother had been delivered.
Chorioamnionitis with plasmolymphocytic in-filtrations and necrotic villi were found in the placenta of one other case (patient 7). Again, in this case, there was no history of maternal genital herpes and the pregnancy was uncomplicated
until the commencement of labor. Because of both failure to progress and fetal distress, cesarean section was performed six hours after amniotomy. The detailed description of the placenta appeared recently in a report by Altshul-er.27
Clinical Course of Disease in Infants
Figure 1 presents the onset and duration of
ara-A treatment, antibacterial therapy, and clinical
signs, including skin vesicles and neurological signs or seizures when present.
Ara-A (10 to 20 mg/kg/day) was given as a continuous intravenous drip for 12 hours daily for ten days. One severely affected infant received the drug for only five days and died. The total dose of ara-A given in all varied from 168 to 720 mg (mean, 397 mg). CNS involvement with increased irritability, alterations in the state of
consciousness from lethargy to coma, reduced oral feedings, vomiting, flaccidity or spasticity of varying degrees were noted in all infants in
SKIN
VESICLES
THROAT
CONJUNCT
IVAE
CSF
WBC
URINE
CERVIX
CORD
BLOOD
I
-
I I I I I I II
AUTOPSY
BRAIN
tJ
AUTOPSY
LIVER
J
AMNIOTIC
FLUID
FECES
(5/9)
(1/12)
(I/Il)
(1/5)
(2/2)
(1/l)
(1/2)
(0/I)
(1/2)
(0/6)
HSV
ISOLATION
FROM
INFANTS
No.of
Patients
with
positive’Positives/Total
no.
cultures
I
of cultures
I
(21/25)
Ii
(18/35)
t
I
:::::::
I
2345678910111213
MATERNAL
FIG. 2. Frequency of herpes simplex virus isolation. Black areas indicate the number ofpatients with isolations and white areas
represent the number of patients from whom no viral isolation was successful.
All infants with CNS involvement demon-strated in the CSF pleocytosis (36 to 1,350 WBC/ cu mm) and protein elevations (140 to 1,400 mg/
100 ml).
Treatment was initiated from three to eight
days after the eruption of the skin vesicles which
appeared to be the hallmark of neonatal HSV
infection in all three groups. However,
nonspe-cific neurologic signs preceded the appearance of
Hct
(%)
WBC(x
I 5 10 15
50
-45
40
35
30
25
16
14
12
l0
8
6
4
3
2
0
TREATMENT
DAY
FIG. 3. Toxicity data from 13 infants show reduction of the arithmetic means of hematocrit and white blood cell count.
Reticulocyte counts do not change.
the only manifestation of disease in two additional infants (8 and 9). As a result, these cases as well as case 5 were misdiagnosed as neonatal sepsis with meningitis and the infants received antibiotic therapy for an average of ten days (5 to 20 days) before the correct diagnosis was made. This contrasted with patients in group I who received antibiotics for only one to three days before the correct diagnosis was made.
One infant in group II developed severe brain damage with a flat EEG. Disseminated intravas-cular coagulation was diagnosed in another patient, and in two additional infants, diabetes insipidus. Four of the five infants in this group subsequently died; the one survivor had micro-cephaly.
In contrast, all four infants in group I, due to
the early appearance of skin vesicles as their initial sign of disease, were treated with ara-A early, within three days (average, 2#{189}days) after the onset of neurologic symptoms and survived. No gross neurologic deficits were noted at 6 months and 1 year of age.
Similarly, all four of the infants in group III with localized infection of the skin and/or eyes survived. One infant demonstrated a residual dendritic ulcer of the cornea.
The skin vesicles in all infants became dry within four days after initiation of therapy, but recurred in two infants weeks after cessation of therapy.
Virology
HSV
was isolated from skin vesicles of 1 1 of 13 infants. The remaining two infants did not have skin vesicles, but HSV-2 was grown from the CSF from one and the throat from the other.Figure 2 shows the sites of positive virus isolation in relation to the number of infants who had cultures obtained from that site
(at left).
Thenumber of attempts made to isolate HSV from various sites in serial cultures obtained to deter-mine the duration of viral persistence at that site are also shown
(at right).
Excretion of HSV from the throat was prolonged and lasted for 6 to 30 days in four infants. This correlates well with the better overall yield of virus from the throat. Suppression of HSV excretion from the throats of three infants was demonstrated by serial quantification of the throat swab material (Table I).
Eleven of 13 viral isolates were serotyped by
indirect immunofluorescent technique26; seven were found to be HSV-2 and four were HSV-1.
Toxicity
Within the dosage of ara-A employed, there was no clinical evidence of toxicity. Serial moni-toring of hepatorenal function profiles and peripheral blood elements showed no changes except for trends of reduction of the arithmetic means of hematocrit and white blood cell counts (Fig. 3). Hematopoiesis is normally reduced during the neonatal period and these changes are probably of no clinical significance as witnessed by normal to elevated reticulocyte counts.
DISCUSSION
It
is well appreciated that the efficacy of any antimicrobial drug is generally better whenfor infants treated with ara-A in group I whose
disease was diagnosed earlier than those in group II. The outcome of infants who were treated late
are predictably and uniformly poor as CNS hemorrhagic necrosis due to HSV infection is already well established.
It is not possible to be certain that the few
infants with localized skin infections would have progressed to disseminated or CNS disease
without treatment. A recent review of 61 patients with neonatal herpes in whom skin lesions were the first manifestation of herpetic disease shows that approximately one half would develop progressive disease.2
Early diagnosis of neonatal HSV infection may pose significant difficulties. Skin vesicles are the characteristic sign of the infection, but are absent
in many cases of neonatal herpes. Besides the fact that they are commonly misdiagnosed for other skin lesions in newborns, an additional problem is that skin vesicles may not appear until other signs of documented CNS involvement have occurred.
These problems were particularly noted in those infants in group II (Fig. 1).
To prevent intrapartum acquired HSV infec-tion, massive screening of near-term pregnant women for genital HSV infection by cytological and/or virological methods may prove helpful to segregate out a manageable group of mothers for
closer observation or for possible cesarean
section. By cytological or virological surveys, the rate of genital HSV infection in lower socioeco-nomic populations of pregnant women has been approximately 1%.2 This rate is about three times as high as that found in nonpregnant women and
is approximately three times less in women of the same socioeconomic group at term.229-3#{176} How-ever, in view of this low incidence, it is generally impractical to culture routinely the genital tracts of all pregnant females for HSV.31 Other than in women demonstrating cytological findings of HSV on random Papanicolaou smear obtained during prenatal visits, the diagnosis of active maternal HSV infection currently depends mainly
on clinical manifestations which occur in less than one third of infected pregnant women and which
are often misdiagnosed.2 Consequently, possible approaches to preventing neonatal exposure to HSV have been limited and treatment of mdi-vidual infants with established HSV infection, although an experimental approach, is often warranted. Perhaps, with the availability of more effective and less toxic antiviral compounds in the
future, more frequent and vigorous application of diagnostic methods can be justified.
The use of antiviral chemotherapeutics
includ-ing IDU and ara-C to treat infants with HSV infection has led to mixed results.7
Ara-C, an antileukemic agent with antiviral activity against DNA-containing viruses, is widely available and has already been used frequently in other DNA viral infections. Due to the acute toxicity of IDU and ara-C to the bone marrow and host immunological system, they have been administered only when the disease is severe and life-threatening or, in reality, late in the course of the illness. Since rapid brain growth is expected during the first two years of extrauterine life in man, any potent inhibitors of DNA synthesis must be cautiously employed for therapy especially in infants. In this study, considering the potential devastating effect of HSV infection on the CNS and the tendency of the infection to disseminate, ara-A, a compound with much less acute toxici-ty32 than IDU and ara-C was administered.
The purpose of the present phase 2 study was to determine the possible antiviral dose range of ara-A and its safety. The results suggested that ara-A, within its nontoxic dose range, may be efficacious in the treatment of neonatal HSV infection provided the drug is given early in the disease course.
However, as past experience has made clear, neonatal HSV infection does not follow a
stereo-typic clinical course with an uniformly fatal outcome. It encompasses a broad spectrum of disease with varying severity ranging from isolated and seemingly harmless vesicular skin eruptions to severe disseminated and often fatal
illness.’ Not only do infants usually recover from localized HSV infections but, in addition, they may spontaneously become well after docu-mented CNS involvement.” It is hard to select, at least at the earliest stage of the disease, candidates for therapy. Also, due to its sometimes unpredictable course, one must be extremely
careful in the evaluation of drug efficacy. It cannot be overemphasized that this type of anti-viral chemotherapy must remain experimental at this stage since the possible long-term side effects of ara-A are not yet clearly defined.
In order to evaluate the true effect of ara-A on the natural course of neonatal HSV infection, a
sufficient number of patients must be studied. A large scale multi-institutional study with a double-blind protocol is now in progress.
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ACKNOWLEDGMENTS
We thank Dr. K. C. Stubbs, Mr. R. Reynolds, and Mr. S. Smith for technical assistance and Mrs. Jan Holley for
secretarial assistance. The following physicians contributed
neonatal cases: Dr. Richard Alper, Minneapolis, Minn.; Drs.
David Carver and Susan Shurin, Johns Hopkins University,
Baltimore, Md.; Drs. Anthony B Minnefor and Richard H.
Rapkin, New Jersey Medical School, Newark, New Jersey; and Drs. Robert H. Parrott and Maureen C. Edwards,
Children’s Hospital National Medical Center, Washington,
D. C. We also thank Drs. Robert W. Chambers and Debora
E. Powell, Ceorgetown University, and Dr. Hynn W. Kim,
Children’s Hospital National Medical Center, Washington,
D. C., for excellent assistance; and Dr. Robert A. Brackett
and Ms. Sylvia Weiss at Parke Davis and Company, Detroit,
Michigan, for excellent laboratory support.
ILLEGITIMATE ISOLATION
Any attempt to analyze the impact of health or education measures without taking other policy measures into consideration involves the logical fallacy of illegitimate isolation.
. . . G. MYRDAL
