Abstract
purpose. Herpetic keratitis is a common sequel of a corneal infection with
herpes simplex virus (HSV)-1. Recrudescent herpetic keratitis (RHK) may
result in irreversible damage to the cornea. Recurrences may be caused
by reactivation of endogenous HSV-1 or reinfection with exogenous
HSV-1. The objective of this study was to determine the incidence and
risk factors involved of HSV-1 superinfection in patients with RHK.
methods. From 30 patients with RHK, sequential corneal HSV-1 isolates were
genotyped by PCR amplification of the hypervariable regions located
within the HSV-1 genes US1, US10/11, and US12. The clinical data from the patients obtained
retrospectively were: ophthalmologic history, clinical picture during
recurrences, number and time points of penetrating keratoplasty (PKP),
and steroid or acyclovir treatment.
results. Whereas the sequential corneal HSV-1 isolates of 19 (63%) of 30
patients had the same genotype (designated as group 1), the sequential
isolates of 11 patients (37%) were genetically different (designated
as group 2). Among the clinical data analyzed, only the time point of
PKP was significantly different between the patient groups. Although no
patients in group 1 had undergone transplantation between samplings, 4
of 11 patients in group 2 underwent PKP during the inter-recurrence
period in the same eye from which the corneal HSV-1 isolates were
obtained.
conclusions. The data demonstrate that RHK is frequently associated with corneal
reinfection with a different HSV-1 strain and suggest that PKP is a
risk factor for corneal HSV-1 superinfection.
Herpes simplex virus (HSV) infections may elicit a variety
of serious diseases in humans, including chronic herpetic
keratitis.
1 2 A hallmark of HSV and other neurotropic
herpes viruses is their ability to establish latency in sensory nerve
ganglia of the host.
1 Despite the induction of an acquired
state of immunity after primary HSV infection, recrudescent herpetic
lesions are often observed.
1 Patients who have had corneal
HSV-1 infection risk recurrent corneal disease throughout life.
Particularly prolonged or recurrent episodes of herpetic keratitis can
result in decreased vision or blindness due to the development of
herpetic stromal keratitis (HSK).
2 3
Recrudescent HSV infections are thought to result from reactivation of
the HSV strain acquired during primary
infection.
4 5 6 However, reinfection with a new HSV
strain (i.e., superinfection) at the site of primary
infection has also been documented.
6 7 The route or mode
of HSV superinfection and its clinical consequences remain enigmatic.
Genetically different HSV strains have been shown to induce different
types of ocular lesions.
8 Furthermore, newly acquired
herpetic keratitis may develop after penetrating keratoplasty (PKP) in
patients who undergo transplantation for reasons unrelated to HSV
infection, suggesting the possibility of HSV-1 transmission through
cornea transplantation.
9 These issues underline the
clinical importance of knowing whether recurrent corneal HSV-1
infections are caused by reactivation of latent virus or superinfection
with a different virus strain. Molecular analyses of corneal HSV-1
isolates may allow distinction between both options.
The genome of HSV-1 consists of a unique long
(U
L) and a unique short
(U
S) component, each of which is flanked by a
pair of oppositely oriented repeat elements. Several hypervariable
regions have been identified in the HSV-1 genome. These regions
encompass unique tandemly repeated sequences, reiterations (Re) that
vary in copy number and nucleotide sequences
(Fig. 1) .
1 10 11 Generally, two types of restriction fragment
length polymorphism (RFLP) analyses are used to differentiate HSV-1
isolates. One type is the variation due mostly to a gain or loss of a
restriction enzyme cleavage site. The other appears as variation in
length of cleaved fragments derived from Re-containing genomic HSV-1
regions.
11 Among the eight Re regions described for HSV-1,
ReIV and -VIII (both located within the introns of genes
US1 and
US12) and ReVII (located within the protein coding
region of genes
US10 and
US11) have been shown to
remain stable during in vitro culture and have been used as sensitive
and reliable markers to differentiate HSV-1 strains.
12 13 14 15
We have recently developed a PCR method, based on the stability and
strain-to-strain differences of ReIV, -VII, and -VIII that has
facilitated the differentiation of up to 92% of unrelated HSV-1
strains.
12 15 The purpose of the present study was to
determine the incidence and risk factors involved in corneal HSV-1
superinfection in patients with recrudescent herpetic keratitis (RHK).
Corneal swab specimens were obtained for diagnostic reasons from
suspected herpetic corneal lesions and were used to inoculate human
embryonic lung fibroblasts. Virus was harvested when approximately 75%
of the monolayer showed cytopathic effect and was subsequently typed
for HSV-1 or -2 by immunocytology and PCR.
15 Serial
samples from 30 immunocompetent patients with recurrent corneal HSV
infections were found in a databank of 408 frozen corneal HSV-1
cultures collected since 1980 at the Rotterdam Eye Hospital (Rotterdam,
The Netherlands). The clinical items scored retrospectively were
anatomic location (i.e., left or right eye), previous history of ocular
disease, clinical picture at presentation of each recurrence, therapy
regimen preceding the culture dates, total number of PKPs, and PKPs
between virus culture dates. The classification of herpetic keratitis
was defined on clinical criteria.
2 The present study was
performed according to the Declaration of Helsinki, and informed
consent was obtained.
Genotypic analyses of the viral strains were performed by
amplification of the hypervariable regions within the HSV-1 genes
US1,
US10/
11, and
US12.
This method is based on strain-to-strain differences in the number of
Re and point mutations within these hypervariable genomic
regions.
10 12 13 15 DNA was extracted from the primary
corneal HSV-1 cultures, lysed in a guanidine isothiocyanate buffer
using a silica solution (Celite; Jansen Chemika, Beers,
Belgium), as described previously.
15 The PCR primers
and conditions for amplifying and detecting by Southern blot analysis
of the hypervariable regions of the HSV-1 genes
US1,
US10/
11, and
US12 have been
described.
15 In case of small differences in length
between amplicons (i.e., PCR products) from individual samples, the PCR
products were run on denaturating (8 M urea) 6% acrylamide gels. The
lengths of the amplicons were estimated by comparison to a 100- and
25-bp DNA ladder (Gibco BRL, Grand Island, NY). To confirm similarities
or differences in amplicon length, all samples were finally
electrophoresed in order of increasing length.
The statistical evaluation of the results was performed using
the Fisher exact test. Results were considered statistically
significant at P < 0.05.
Patients’ Characteristics and Genotypic Analyses of
Sequential Corneal HSV-1 Isolates
The group of 30 patients with RHK included in this study
consisted of 13 women and 17 men (mean age, 58.1 years; range, 17–78).
From each patient, two (
n = 25) or three
(
n = 4) sequential corneal HSV-1 isolates were obtained
(mean time interval, 29.8 months; range, 0–170). Patient 22
had bilateral herpetic keratitis
(Table 1) .
To differentiate whether RHK is due to reactivation of latent HSV-1 or
superinfection with another HSV-1 strain, the sequential corneal HSV-1
isolates of the patients with RHK were genotyped using a recently
developed PCR-based DNA fingerprint assay.
15 The results
of the PCR analyses, on the hypervariable regions of the genes
US1,
US10/
11, and
US12,
performed on the corneal HSV-1 isolates are summarized in
Table 1 . As
an example, the size fractionation and Southern blot analyses of the
US1- and
US12-specific amplicons obtained from
the sequential samples of patients 1 through 5 and 20 through 24 are
shown in
Figure 2 . The sequential corneal HSV-1 isolates of 19 (63%) of the 30 patients
and 11 (37%) of the 30 patients showed either identical (patients
1–19; designated patient group 1) or different genotypes (patients
20–30; designated patient group 2), respectively
(Table 1) . The data
suggest that more than one third of the corneas of the patients with
RHK were superinfected with a different HSV-1 strain. In the case of
patient 30, the newly acquired HSV-1 strain was cultured pending two
post-PKP recurrences. This suggests that the newly acquired HSV-1
strain had colonized the recipient. Combining the results of the three
amplified genomic regions showed that the majority of the
distinguishable HSV-1 isolates displayed unique combinations of
amplicons
(Table 1) .
In the case of patient 22, the data indicated that the bilateral
herpetic keratitis was due to infections with different HSV-1 strains
in either cornea. Patient 30 had two different HSV-1 strains
identified. In the third episode sampled, the strain identified during
the second recurrence was isolated
(Table 1) .
Comparison of Clinical Characteristics of Patients with RHK in
Patient Groups 1 and 2
Compared with previous reports on patients with
RHK,
2 9 our cohort consisted mainly of patients with
severe entities of HSV-induced keratitis, such as herpetic stromal and
necrotizing keratitis. This is also reflected in the high number of
PKPs in the patient cohort (
Table 2 ; mean PKPs, 1.4 per patient; range, 0–6).
The clinical characteristics of the patients in groups 1 and 2 were
compared, to identify the factors predisposing for corneal HSV-1
superinfection. Overall, the immune status and ophthalmic condition did
not differ significantly between both groups (data not shown).
Additionally, gender, inter-recurrence period, anatomic location
of the lesions (left or right eye), ocular history, and clinical
picture at time of recurrences were not statistically different between
both groups
(Tables 1 2) .
The clinical outcome of corticosteroid treatment before or during
the convalescence period was not statistically different between both
groups. The potential effects of long-term (val)acyclovir treatment
were not numerous enough to be interpreted (data not shown).
Although the mean number of PKP per patient did not significantly
differ between both groups, indicating that both groups were comparable
in disease severity, a correlation between corneal HSV-1 superinfection
and time point of PKP was observed. Whereas no patient in group 1
received a corneal transplant between the sampled recurrences, 4 of the
11 patients in group 2 underwent a PKP during the inter-recurrence
period in the same eye from which the sequential corneal HSV-1 isolates
were obtained (
Table 2 ;
P = 0.012). Patient 30 received
a corneal allograft between the first and second sampled recurrence.
HSVs have the ability to reside in latent form within neurons of
the sensory ganglia that innervate the initial site of infection. It is
therefore assumed that recurrent herpetic lesions are due to
reactivation of the HSV strain acquired during the primary
infection.
1 4 5 6 In contrast, HSV superinfection in
patients with recrudescent herpetic lesions has been
documented.
6 7 Patients with recurrent herpetic keratitis
risk the development of HSK, a leading cause of corneal blindness
worldwide.
2 3 The objective of the present study was to
examine the two types of origins and risk factors involved in corneal
HSV-1 superinfection in 30 patients with HSV-1–induced RHK.
Genotypic analyses of sequential corneal HSV-1 isolates from 30
patients with RHK demonstrated that 63% of the patients (patients
1–19; designated as group 1) had evidence of reactivation of the same
HSV-1 strain. From five patients in group 1, the isolates were obtained
from separate eyes. HSV-1 infection of the contralateral cornea most
likely occurred through the external route (cross-infection). It was
interesting that sequential isolates of 37% of the patients (patients
20–30; designated as group 2) had a different genotype, suggesting
corneal HSV-1 superinfection in the inter-recurrence period.
Alternatively, the instability of the analyzed hypervariable regions
may account for these differences. HSVs, similar to other DNA viruses,
have less genomic variability than RNA viruses and are genetically more
stable after in vitro passages.
11 15 In addition to
standard RFLP, several hypervariable regions within the HSV-1 genome
have been used to differentiate HSV-1 isolates
genetically.
11 Intratypic variation of the regions results
from differences in the number of Re and point
mutations.
10 12 13 The stability of the eight
HSV-1–specific Re regions described varies extensively.
11 Genotypic analyses of HSV-1 single-plaque clones compared with their
parental strain have shown that the hypervariable regions located
within the HSV-1 genes
US1,
US12, and
US10/11 remain stable during in vitro
culture.
13 15 Moreover, the mean inter-recurrence period
of patient group 1 (30.4 months) and the proofreading activity of
Pfu DNA polymerase, implies that the intraindividual HSV-1
genotype differences are most likely not due to a genetic alteration of
the initial strain or errors in amplifying these highly GC-rich DNA
sequences, respectively.
Analogous to our study, reinfection with new HSV-2 strains has been
described in two of three patients with recurrent HSV-2 genital
herpes.
7 The latter study and our data indicate that HSV
superinfection is not as rare as previously
suggested.
4 5 6 To differentiate HSV strains, most
investigators have used RFLP analyses with 6-bp recognizing restriction
enzymes (REs).
4 5 6 The lower efficacy of 6-bp RE, compared
with the 4-bp RE, to differentiate HSV-1 strains may account for the
different frequencies of HSV superinfection described.
11
Generally, corneal HSV-1 infection results in the development of
herpetic epithelial keratitis in approximately two thirds of
patients.
2 In the present study, however, the patient
cohort consisted predominantly of patients with severe entities of
herpetic keratitis
(Table 2) . Selection of individuals with a higher
susceptibility for corneal HSV-1 infection may have occurred.
Alternatively, the patients in group 2 may have been superinfected with
a more virulent HSV-1 strain.
Among the clinical data analyzed, only the time point of PKP was
significantly different between the patient groups. Although no
patients in group 1 had undergone transplantation between sampling, 4
of 11 patients in group 2 underwent PKP during the inter-recurrence
period in the same eye from which the corneal HSV-1 isolates were
obtained. The data suggest that PKP is a risk factor for corneal HSV-1
superinfection. Primary graft failure and endothelial abnormalities of
cultured eye bank corneas have been associated with the presence of
HSV-1 DNA in affected corneal allografts.
16 The high
prevalence of HSV-1 DNA in eye bank corneas (∼ 10%)
16 has led to the hypothesis of HSV-1 latency in corneas. Although
expression of HSV-1 latency-associated transcript, a marker of latency,
has been detected in latently infected rabbit corneas and human HSK
corneas, corneal HSV-1 latency remains controversial.
16 17 Recently, Zheng et al.
18 have demonstrated HSV-1
transmission through PKP in an experimental rabbit model. HSV-1 DNA was
detected in recipient corneal rims and the innervating trigeminal
ganglion (TG) of naive rabbits that received corneal allografts
from latently infected rabbits. Moreover, infectious HSV-1 was
recovered from the tear film of the rabbits that had undergone
transplantation.
18 Besides true ocular viral latency,
putative HSV-1 transmission through PKP may be due to coincidental
shedding of small amounts of infectious virus from the allograft or a
low level of viral replication in corneal resident cells in the
allograft at time of PKP.
18 19
Alternatively, the TG may harbor a mixture of HSV-1 strains with which
the patients were previously latently infected, before PKP. In animal
model studies, corneal trauma (similar to PKP) has been shown to induce
reactivation of HSV-1 causing corneal HSV-1
infection.
20 21 Assuming that the human TG can be latently
infected with multiple HSV-1 strains, PKP may serve as a powerful
reactivation stimulus to certain portions of the TG, allowing multiple
strains to reactivate.
22
In conclusion, this study is the first to demonstrate a high frequency
of corneal HSV-1 superinfection in patients with RHK. Although we could
not determine the source or mode of corneal HSV-1 superinfection in
patient group 2, the data suggest that PKP may be a risk factor for
transmission of HSV-1 with subsequent reactivation of the donor-derived
HSV-1 strain in the corneal allograft. Recently, we have genetically
characterized HSV-1 DNA isolated from a donor cornea before and after
PKP in a patient with newly acquired herpetic keratitis. The DNA
sequences were identical in both strains, providing conclusive evidence
for graft-to-host transmission of HSV-1 through corneal
allograft.
23