MAJOR ARTICLE

Antiviral Susceptibility of Herpes Simplex

Viruses and Its Clinical Correlates: A Single
Centers Experience
N. Rabella,1 M. Otegui,1 R. Labeaga,1 P. Rodrguez,1 N. Margall,1 M. Gurgu,2 and G. Prats1
1
Servei de Microbiologia and 2Unitat de Malalties Infeccioses, Hospital de la Santa Creu i Sant Pau, Universitat Auto`noma de Barcelona, Spain

The in vitro susceptibility to acyclovir of 204 herpes simplex virus isolates from 165 immunocompromised
patients treated at our hospital was determined by the cytopathic effect reduction assay. Approximately 95%
of herpes simplex virus 1 and 73% of herpes simplex virus 2 isolates were inhibited by acyclovir at concen-
trations of !2 mg/mL. From 8 patients (5%), an isolate with low susceptibility to acyclovir (50% inhibitory
dose, 13 mg/mL) was recovered. Medical records of 83 patients were reviewed. Lesions resolved in most of the
patients, independent of treatment. Treatment failures were not always associated with isolation of an in
vitroresistant virus. On the contrary, when a virus with low susceptibility to acyclovir was isolated, resolution
of the lesion was the rule. In 9 of 10 patients with subsequent recurrent episodes of disease, the susceptibility
of the viruses isolated was similar to that of the first episode. Routine susceptibility testing in our geographic
area is not encouraged because of the low incidence of acyclovir-resistant herpes simplex viruses.

Infections caused by herpes simplex virus (HSV) are a
major cause of morbidity in immunosuppressed hosts,
such as patients who are positive for HIV or are un-
dergoing organ transplantation. Acyclovir (Acv) is the
drug of choice for treatment of these patients. Admin-
istered orally or intravenously, it decreases pain and
viral shedding and accelerates healing [1]. Topical ther-
apy can also be useful for these patients, although it is
not as effective [2]. Mucocutaneous HSV infections are
usually self-limited in the immunocompetent host. Be-
cause immunosuppressed patients cannot depend on
their immune system to eliminate the virus, infection
can result in extensive and persistent ulcerative disease
with continuous viral shedding and lack of response to
Received 9 July 2001; revised 27 November 2001; electronically published 11
March 2002.
Financial support: Partially support from the Fondo de Investigaciones Sanitarias
de la Seguridad Social (FISS), Spain (grant 93/0405). Spanish Ministerio de
Educacion y Cultura (grant AP94 35775298 [to M.O.]).
Reprints or correspondence: Dr. Nuria Rabella, Av. Sant Antoni Ma Claret 167,
08025 Barcelona, Spain (nrabella@hsp.santpau.es).
Clinical Infectious Diseases 2002; 34:105560
 2002 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2002/3408-0004$03.00
prolonged Acv therapy; strains of HSV that are less
susceptible or resistant to Acv have occasionally been
recovered from these patients [3]. Otherwise, in im-
munocompromised patients who receive Acv for the
management of acute HSV disease, the incidence of
resistance is extremely low. In addition, Acv-resistant
strains of HSV have rarely been isolated from immu-
nocompetent patients [4].
The use of susceptibility testing could help improve
the management of patients with herpesvirus disease
who are unresponsive to standard regimens of Acv. Pa-
tients whose lesions persist or worsen while they are
receiving Acv therapy could benefit from susceptibility
studies. In such cases, knowledge of a strains in vitro
susceptibility to Acv would be useful for the selection
of alternative antiviral therapy [57], which might in-
volve increasing the dose of oral Acv or changing to
high-dose continuous infusion of Acv (for those pa-
tients who do not respond to standard regimens of Acv)
[8, 9]. To determine the susceptibility of HSV to Acv,
various tests have been designed. The most frequently
used tests are the plaque reduction assay (PRA), the
viral cytopathic effect reduction assay (CRA), the dye-

HSV Antiviral Susceptibility CID 2002:34 (15 April) 1055

uptake method (DUA), and the DNA hybridization assay.
There is no consensus regarding the level of in vitro suscep-
tibility that indicates a drug-resistant virus, but many different
types of assays have shown that Acv-resistant isolates are usually
susceptible to an Acv concentration of 123 mg/mL [1012].
Nevertheless, correlation between clinical findings and in vitro
susceptibility to Acv has not been definitely established, and
further studies are needed. The present article reports the results
of susceptibility testing of HSV isolates from immunocom-
promised patients who attended in a university tertiary-care
hospital and provides further data on the clinical correlates to
the in vitro results.
PATIENTS, MATERIALS, AND METHODS
Patients and virus isolation techniques. A total of 204 iso-
lates (131 HSV-1 isolates and 73 HSV-2 isolates) were recovered
from 165 immunosuppressed patients (89 patients who had
AIDS, 18 who had hematological malignancies, 20 who had
received heart transplants, 8 who had received bone marrow
transplants, 14 who were receiving corticoid therapy, 10 who
had neoplastic disease, 4 who had received kidney transplants,
1 who had common variable hypogammaglobulinemia, and 1
who had diabetes). HSV strains were isolated from 178 mu-
cocutaneous lesions (70 oral, 4 pharyngeal, 8 nasal, 16 labial,
25 genital, 34 anal, and 21 cutaneous exudates), 19 specimens
of bronchoalveolar lavage [BAL] fluid, 4 esophageal biopsy
specimens, 1 brain biopsy specimen, 1 rectal biopsy specimen,
and 1 urine specimen. For isolation of HSV, specimens for
culture were routinely obtained by swabbing the base of mu-
cosal or cutaneous ulcers. The swabs or specimens were placed
in transport medium and inoculated on cell monolayers, usu-
ally Vero or HeLa cells. BAL fluid and biopsy specimens were
transported to the laboratory without use of transport medium.
If 2 specimens were available for the same episode of disease,
both were used.
Virus isolation, identification, and typing (with use of the
Microtrak HSV-1/HSV-2 culture confirmation/typing test;
Syva) were performed by using conventional techniques [13].
Viruses were then passaged in Vero cells to produce a working
stock with a known titer [14] and were stored at 80C until
susceptibility tests were performed.
Cytopathic effect reduction assay. In vitro susceptibility
of HSV isolates to Acv was determined by the cytopathic effect
reduction assay, performed in 96-well plates. In summary, Vero
cells were cultured and maintained in Eagles minimum essen-
tial medium supplemented with nonessential amino acids, l-
glutamine, and fetal calf serum. Cell cultures were infected with
100 TCID50 (range, 31.6316 TCID50) of each virus, and serial
2-fold dilutions of Acv (range, 0.0250 mg/mL) were added to
the wells. After incubation for 72 h, the cytopathic effect of
1056 CID 2002:34 (15 April) Rabella et al.
each strain was recorded and scored as positive or negative.
We considered a well result to be positive if there was 1 focal
lesion of cytopathic effect. The Karber method [15] was used
to determine the drug concentration causing a 50% reduction
in viral cytopathic effect, or 50% inhibitory dose (ID50) and
the 90% inhibitory dose (ID90) of Acvthat is, the dilution at
which 50% or 90% of the inoculated cultures had negative
results. The results are expressed as micrograms per milliliter
[16].
One resistant strain derived by repeated passage in the pres-
ence of Acv was introduced in the study as a control (kindly
provided by Dr. F. Salmeron, Centro Nacional de Microbio-
loga, Virologa e Inmunologa Sanitarias de Majadahonda,
Spain). Control wells that exclusively contained either cells with
virus or the higher concentration of Acv (50 mg/mL) were
included in each assay. Of the 165 patients, medical records
were available for 83, who had a total of 96 episodes of HSV
disease. Treatment and evolution of the lesions were reviewed.
RESULTS
Antiviral susceptibility. The in vitro Acv susceptibilities of
204 HSV clinical isolates recovered from 165 patients were
determined. The median ID50 of Acv for HSV-1 was 0.615 mg/
mL (range, 0.0504.456 mg/mL). For HSV-2, the median ID50
was 1.698 mg/mL (range, 0.4725.296 mg/mL).
Approximately 86% of HSV-1 isolates were inhibited by Acv
concentrations of !1 mg/mL, and 195% were inhibited by con-
centrations of !2 mg/mL. A total of 73% of HSV-2 isolates were
inhibited by Acv concentrations of !2 mg/mL. Of 204 virus
strains studied, 9 (4%; 1 strain of HSV-1 and 8 strains of HSV-
2) showed susceptibility to Acv at an ID50 of 13 mg/mL.
Although a wide spectrum of susceptibilities was found
among the HSV strains studied, none of the strains had ID50
values of Acv comparable to that of the resistant control strain
(ID50, 12.559 mg/mL). For 6 patients from whom 2 strains were
recovered from the same episode of disease, the Acv ID50 values
were very similar (table 1). All the strains isolated from lesions
other than mucocutaneous lesions had Acv ID50 values of !3
mg/mL.
Clinical response. We recorded data on the treatment and
evolution of 96 episodes of HSV disease in 83 patients. Acv
therapy was administered for 90 episodes. In 46 cases, the treat-
ment was oral; in 15 cases, intravenous; and, in 11 cases, topical.
In 18 cases, the route of administration was combined (oral
and/or intravenous and/or topical). The lesions resolved in 84
cases. For 6 patients, lesions did not resolve, despite antiviral
therapy. Four of these patients had incomplete treatment and
died of their disease within a few days (1 had received a bone
marrow transplant, 2 had lymphoma, and 1 had AIDS); these
patients were excluded from the analysis. The other 2 patients
 Table 1. Susceptibility values for herpes simplex the evolution of the episodes of disease are shown in table 2.
virus (HSV) isolates recovered during single epi- Available clinical data concerning individual patients from
sodes of HSV disease.
whom isolates were recovered with Acv ID50 values of 12 mg/
Acv ID50
mL are shown in table 3. All these patients lesions resolved.
of isolate, Time second Recurrences of disease after therapy. There were 15 re-
a
Patient Isolate mg/mL specimen obtained currences in 10 patients (the episodes of disease were separated
2 HSV-1 0.084 by 0.523 months). Of 27 strains of HSV isolated, only 1 had
0.199 Same day an elevated Acv ID50 (table 4). Patient 41 had 4 episodes during
6 HSV-1 0.334 the study period; in the third episode, a resistant HSV-2 strain
0.334 2 days later was isolated, and, 50 days after that episode resolved without
12 HSV-2 1.330 treatment, a susceptible HSV strain was isolated.
0.941 1 day later
17 HSV-1 0.281
0.941 Same day DISCUSSION
31 HSV-1 0.236
The clinical development of antiviral drug resistance has been
0.561 Same day
increasingly reported for infections caused by HSV [17]. One
92 HSV-1 0.334
of the main problems in testing for in vitro susceptibility is
0.472 1 day later
that antiviral studies are not well standardized. To determine
NOTE. Acv, acyclovir; ID50, drug concentration causing a the Acv susceptibility of HSV, various tests have been designed,
50% reduction in viral cytopathic effect. There were no special
reasons for obtaining 2 samples from the same lesion. such as the PRA, the CRA, the DUA, and the DNA hybridi-
a
Time when the second specimens were obtained relative zation assay.
to when the first specimen was obtained. ID50 values obtained by the hybridization method correlate
well with values obtained by the PRA [18]. The DUA yields
received prolonged treatment, and their lesions improved but ID50 values that are 510-fold higher than the values determined
did not heal completely. HSV isolates recovered from these by the PRA [10]. Testing of a single virus strain by these dif-
lesions were susceptible to Acv (ID50, 0.941 and 1.330 mg/mL). ferent assays may produce different results. Variables that affect
Both patients died 14 months later, because of the natural the results obtained by each of the assays include the inoculum
progression of their disease (AIDS). effect, the media used, and the conditions of incubation [19].
Six patients received no antiviral treatment and their lesions Even with a single type of assay, results are extremely dependent
resolved spontaneously. Of 79 patients with data available for on the origin of the cell culture used [20]. In the case of Acv,
analysis, 2 (patients 5 and 45) had persistence of lesions and intracellular levels of endogenous thymidine may be responsible
failure to eradicate the virus, and they were followed for 2.5 for such differences [21].
and 1.5 months, respectively. The lesions of patient 5 responded PRA has been considered by some investigators to be the
to Acv when treatment was initiated, and the lesions of patient reference method for in vitro study of susceptibility to anti-
45 responded to Acv when the doses were increased. For these virals, but the fact that HSV is a rapidly growing virus and has
2 patients, the in vitro susceptibility of HSV isolated at different a clear-cut cell-killing effect allows for the use of easier methods
times and from different sites, the treatment administered, and for the study of these viruses. The CRA was introduced by Ho

Table 2. Susceptibility of herpes simplex virus (HSV) isolates recovered from 2 patients with persistent lesions.

Date Acv ID50 Acv ID90 Type and

Underlying CD4 count, Site of specimen of isolate, of isolate, duration of
Patient disease cells/mm3 lesion Isolate obtained mg/mL mg/mL Acv therapya Lesion outcome
5 AIDS !100 Nasal HSV-1 11 Feb 0.397 0.69 None Persistence
12 Mar 0.334 0.60 None Persistence
26 Apr 0.472 0.72 Oral, 14 days; Resolution
topical, 9 days
45 AIDS 70 Perianal HSV-2 25 Jul 0.941 2.40 Oral, 14 days Persistence
23 Aug 0.792 1.38 Oral, 37 daysb Persistence
5 Sep 1.119 2.75 Resolution

NOTE. Acv, acyclovir; ID50, drug concentration causing a 50% reduction in viral cytopathic effect; ID90, drug concentration causing a 90% reduction in viral
cytopathic effect.
a
Duration of therapy is measured from the date the specimen was obtained. The oral dosage of Acv was 200 mg given 5 times per day.
b
Because of the persistence of lesions, the dosage of Acv was increased to 800 mg given 5 times per day until lesions resolved.

HSV Antiviral Susceptibility CID 2002:34 (15 April) 1057

Table 3. Clinical features of patients with herpes simplex virus (HSV) infections that had low susceptibility to acyclovir treatment.

Underlying Acv ID50 Acv ID90

CD4 count, disease or of isolate, of isolate, Type of
a
Patient cells/mm3 condition Specimen obtained Isolate mg/mL mg/mL Acv therapy Lesion outcome
51 Malignancy Cutaneous exudate HSV-1 2.654 4.78 Oral Resolution
100 54 AIDS Esophageal biopsy HSV-1 2.654 4.78 Intravenous Resolution
b
103 !200 AIDS Oral exudate HSV-1 4.456 9.53 Oral Resolution
c
41 67 AIDS Perianal exudate HSV-2 2.654 5.48 Topical Resolution
41c 13 AIDS Perianal exudate HSV-2 3.155 9.53 None Resolution
61 46 AIDS Perianal exudate HSV-2 2.233 4.78 Oral Resolution
62 Malignancy Perianal exudate HSV-2 2.233 4.78 Topical Resolution
101 45 AIDS Perianal exudate HSV-2 2.654 4.78 Oral Resolution
102 56 AIDS Perianal exudate HSV-2 2.654 4.78 Oral Resolution
b
107 Heart TP Genital exudate HSV-2 4.456 5.88 Oral Resolution
126 Diabetes Cutaneous exudate HSV-2 5.296 19.01 Topical Resolution

NOTE. Acv, acyclovir; ID50, drug concentration causing a 50% reduction in viral cytopathic effect; ID90, drug concentration causing a 90% reduction in viral
cytopathic effect; TP, transplantation.
a
The oral dosage of Acv was 200 mg given 5 times per day for 10 days, and the intravenous dosage of Acv was 400 mg given every 8 h for 10 days.
b
The lesions that yielded these isolates resolved with standard regimens of oral Acv therapy.
c
Two episodes separated by 17 months.

and Enders [22] for the in vitro study of the susceptibility of
cells to interferon. De Clercq et al. [23] and DeClerq [24] used
the CRA to study the susceptibility of HSV to antivirals. The
addition of the vital dye neutral red to the drug-treated, virus-
infected cultures permits automated reading. This method was
named the dye-uptake method. McLaren et al. [25] adapted
the DUA method for the study of HSV susceptibility to acy-
clovir. The DUA is equivalent to the CRA, the only difference
being the reading method, and the ID50 values determined by
the CRA and the DUA are comparable, if the optimal condi-
tions for both assays are applied [26].
In our study, we used the CRA, with Vero cells, to test all
isolates. This assay is based on measurements of the inhibition
of the cytopathic effect and provides a simple determination
of Acv susceptibility [27]. Using this assay, we found that re-
peated testing of several specimens of the same lesion revealed
similar susceptibilities (table 1), which indicates that the assay
has an acceptable degree of reproducibility.
Our results show that the mean ID50 value for HSV-1 (0.615
mg/mL) is almost 3 times lower than that for HSV-2 (1.698 mg/
mL). These differences and the overlapping of values for the 2
types of viruses confirm previous observations [2730].
There is no consensus of opinion regarding which level of
in vitro susceptibility indicates a resistant virus, but most in-
vestigators agree that Acv-resistant isolates are usually suscep-
tible to Acv concentrations of 12 mg/mL. However, Chatis and
Crumpacker [31] pointed out that an Acv ID50 value of 12 mg/
mL is unusually high for the definition of resistance to Acv,
and use of this value could underestimate the real incidence of
resistance. We detected 26 HSV strains (6 HSV-1 strains and
20 HSV-2 strains)or 13% of the strains isolatedthat had
Acv ID50 values of 12 mg/mL. Swierkosz and Biron [32] pro-
posed that when the DUA is used to study susceptibility, the
ID50 value denoting resistance would be 13 mg/mL. In our study,
9 (4%) of 204 virus strains studied (recovered from 5% of the
patients), were resistant at Acv ID50 values of 3 mg/mL.
It should be noted that, although a low susceptibility to Acv
in vitro was detected in the HSV strains in our study, resistance
did not seem to be the major cause of treatment failure, because
other factors, such as the patients immunologic status, may
have contributed to the poor clinical response. Medical records
were available for 10 patients infected with strains of HSV for
which the Acv ID50 was 12 mg/mL at presentation. The lesions
of all of these 10 patients resolved, with or without Acv treat-
ment (table 3).
On the other hand, persistence of lesions in spite of therapy
was unrelated to the Acv ID50 of the infecting strain. Testing
results for the 2 patients (patients 45 and 5) whose lesions
persisted and then finally resolved (table 2) show that all isolates
were susceptible to Acv. Patient 45 received a standard regimen
of Acv, and the HSV isolate was susceptible in vitro. During
treatment, a new lesion appeared at another site (probably be-
cause of autoinoculation), and, from this lesion, we were also
able to isolate a strain of HSV that was susceptible to Acv. The
lesions resolved completely after treatment with higher doses
of Acv. In patient 5, the lesion did not resolve until the patient
received Acv (table 2).
HSV infections are usually mild and self-limited. Cell-me-
diated immunity plays a significant role in the eradication of
HSV disease. The great variability in the abilities of immu-
nosuppressed patients to control viral infections is a major
confounding factor in efficacy testing of any antiviral therapy.

1058 CID 2002:34 (15 April) Rabella et al.

 Table 4. Susceptibility of herpes simplex virus (HSV) isolates recovered during
recurrences of disease in 10 patients.

Episode
(days since Acv ID50
previous of isolate, Type of
Patient Isolate episode) mg/mL Acv therapya Lesion outcome
1 HSV-1 1 (0) 0.397 Oral Resolution
2 (63) 0.199 Oral Resolution
2 HSV-1 1 (0) 0.167 Intravenous Resolution
2 (43) 0.281 Topical Resolution
3 (350) 0.084 Intravenous, oral Resolution
4b HSV-2 1 (0) 0.941 Topical Resolution
HSV-1 2 (146) 0.792 Topical Resolution
5c HSV-1 1 (0) 0.397 None Persistence
2 (29) 0.334 None Persistence
3 (74) 0.472 Oral Resolution
4 (116) 0.167 Oral Resolution
6 HSV-1 1 (0) 0.666 Intravenous Resolution
2 (50) 0.334 Intravenous Resolution
3 (96) 0.141 Intravenous Persistenced
8 HSV-1 1 (0) 0.397 Oral Resolution
2 (398) 0.666 None Resolution
10 HSV-1 1 (0) 0.199 Intravenous Resolution
2 (689) 0.397 Oral Resolution
12b HSV-2 1 (0) 1.330 Intravenous, oral Resolution
HSV-1 2 (528) 0.397 Oral Resolution
40 HSV-2 1 (0) 1.581 Oral Resolution
2 (353) 1.581 Intravenous Resolution
3 (479) 0.941 Oral Persistence
41 HSV-2 1 (0) 2.654 Topical Resolution
2 (123) 0.792 Topical Resolution
3 (498) 3.155 None Resolution
3 (548) 0.561 Oral Resolution

NOTE. Acv, acyclovir; ID50, drug concentration causing a 50% reduction in viral cytopathic
effect.
a
The oral dosage of Acv was 200 mg given 5 times per day for 10 days, and the intravenous
dosage of Acv was 400 mg given every 8 h for 10 days.
b
Recurrence with different virus type at another site.
c
For episodes 1, 2, and 3, isolates were recovered from specimens obtained from the same
lesion on different days; episode 4 was a recurrence.
d
Patient died before completion of treatment.

Favorable responses were observed in most of our patients
regardless of therapy. Six episodes of disease resolved without
treatment. Moreover, in the 3 episodes for which the HSV
isolates were susceptible at Acv ID50 values of 13 mg/mL and
for which the patients were treated with Acv, the lesions re-
solved with the use of standard regimens of the antiviral agent,
a result that reinforces the finding that most HSV infections
are self-limited, independent of therapy.
Refractory disease due to HSV in these patients could be
related to circumstances other than in vitro susceptibility. The
description of HSV as resistant at Acv ID50 values of 13 mg/
mL is probably appropriate, on the basis of multiple studies
[3236], other factors, such as incomplete adherence to therapy,
poor absorption of the drug, and interaction between drugs,
should be taken into account before resistance to antiviral ther-
apy is suspected.
After primary infection, HSV remains latent in the organism
for life, and, from time to time, the virus reactivates and pro-
duces a recurrent disease. Recurrences probably reflect the virus
that caused the primary infection [19]. During follow-up, a
total of 15 recurrences of disease were recorded. All but 1 of
the HSV isolates were clearly susceptible to Acv in vitro, a
finding that shows that the appearance of resistance is very
infrequent when Acv is used at standard dosages to manage
acute infections.
To summarize, in 5% of the patients in our study (4 of 83),

HSV Antiviral Susceptibility CID 2002:34 (15 April) 1059

we detected a strain of HSV with low susceptibility to Acv in
vitro (ID50, 13 mg/mL). The results of CRA had a close correlation
with the clinical responses in our patients; 97.5% of lesions that
yielded strains of HSV that became susceptible at an Acv ID50
of !3 mg/mL resolved with Acv treatment. For 2 patients, CRA
results were unable to predict treatment failure. The immuno-
logic status of these 2 patients (end-stage AIDS) may have played
a significant role in the persistence of the lesions.
In our experience, there is no need to routinely test HSV
isolates for susceptibility to Acv. Determination of the suscep-
tibilities of strains of HSV should be indicated only for viruses
isolated from patients who have severe immunologic distur-
bance and lesions that persist or worsen while they are receiving
Acv therapy.
Acknowledgment
We are grateful to Dr. F. Salmeron of the Centro Nacional
de Microbiologa, Virologa e Inmunologa Sanitarias de Ma-
jadahonda, Spain, for all his help and for supplying the resistant
strain.
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