Diagnosis and Quantitative Detection of Herpes Simplex Virus DNA in Corneal Ulcers
Herpes simplex keratitis is one of the important causes of blindness and its early diagnosis is essential to the treatment of the respective patients accordingly. The aim of this study was to diagnose and analyze quantitatively the herpes simplex DNA in patients with suspected Herpes keratitis using TaqMan real-time PCR method. Corneal swabs from HSV keratitis suspected patients were collected from September 2005 to December 2009. Upon DNA extraction, the samples were analyzed by quantitative TaqMan real-time PCR assay. In this method, a set of primers amplified a common sequence of HSV-1 and HSV-2 glycoprotein B gene. The copy number of unknown samples were expressed via standard curve drawn with known amount of amplified cloned plasmid containing target sequences. Of the 296 samples, 178 (60.1%) belonged to males and 118 (39.9%) to females. The HSV DNA was detected in 111 (37.5%) of the cases by PCR, consisting of 69 males and 42 females. The drawn standard curve was linear in 10 to 106 copies of virus (R2 = 0.982). The ranges of the HSV DNA copy numbers in the collected samples were detected from 2.7x103 to 6.1x105 copies/samples. Present results suggest that Herpes simplex keratitis remains as an epidemiologically important eye disease and improvement in the monitoring and detecting of ocular herpetic disease by quantitative PCR method is informative and helpful to the clinical diagnosis, decision for treatment and follow up.
November 26, 2011; Accepted: January 14, 2012;
Published: February 13, 2012
Herpes Simplex Virus (HSV) keratitis is an important cause of ocular morbidity.
Corneal HSV infections cause number of clinical manifestations ranging from
blepharitis, acute infectious epithelial keratitis to the potentially blinding
chronic inflammatory disease herpetic stromal keratitis (Kaye
and Choudhary, 2006). Following primary infection through the mouth, nasal
cavity and other parts of the facial area including the eyes, HSV may become
latent for life in trigeminal ganglion. Depending on the individuals physiologic
status and environmental conditions including exposure to the sunlight, presence
of stressful and trauma and finally being immuno-compromised, the recurrence
of HSV infection may happen to the individuals. The trigeminal ganglion innervates
the eye that is, the reactivation may also reach the corneal tissues through
anterograde axonal spreading from the trigeminal ganglion (Toma
et al., 2008; Ziyaeyan et al., 2007a).
Frequent recurrences followed by immunological responses may lead to corneal
scarring, thinning, neovascularization (Suryawanshi et
al., 2011) and consequently the patient may need to undergo keratoplasty
(Knickelbein et al., 2009; Inoue,
2008; Carr and Tomanek, 2006).
Herpes Simplex Keratitis (HSK) can be diagnosed by multiple lab studies, of
which the gold standard is the isolation of HSV from the cornea; however, this
technique is time consuming and possesses low level of sensitivity (El-Aal
et al., 2006). Another technique is cytology and detection of intranuclear
inclusions and multinucleated giant cells in the slides of the corneal scrapping.
Although it is faster, it is not sensitive enough (Subhan
et al., 2004). Enzyme or fluorescence based immuno-histochemical
techniques have acceptable sensitivity, but the interpretation of their consequently
results is challenging in most cases and yields false positive results. Polymerase
Chain Reaction (PCR) is among the techniques quickly replacing the previous
ones. It possesses adequate levels of sensitivity and specificity (Soleimanjahi,
et al., 2007; El-Aal et al., 2006;
Amir and Paul, 2006; Subhan et
al., 2004). Quantitative real-time PCR has a high level of sensitivity
and specificity and is time saving, compared to other PCR methods (Fukuda
et al., 2003). In addition, since it reports the DNA copy number
in the involved tissues, it can serve as the direct evidence of infection severity
in corneal tissues (Remeijer et al., 2009). Clinicians
can also monitor the consumption and efficacy of the treatment with anti-viral
In the present study, corneal scraping specimens were used to detect and quantify HSV DNA by the TaqMan quantitative real-time PCR method in the suspected HSK patients.
MATERIALS AND METHODS
Patients and samples collection: Two hundred ninety-six patients clinically
suspected with herpetic corneal infection were enrolled in this study. One hundred
seventy-eight of the patients were male and 118 were female and their ages ranged
from 11 months to 89 years, with a mean of 57.2 years.
The samples consisting of corneal epithelial scrapings in 500 μL of Viral Transport Media (VTM) and were submitted to the Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz, Iran between September 2005 and December 2009 for HSV keratitis diagnosis. The in-house prepared VTM contained Hanks BSS with 1% bovine serum albumin, sodium bicarbonate, phenol red, penicillin and gentamicin. Swab samples were stored at -20°C until DNA extraction. The study was performed based on Declaration of Helsinki protocol and approved by the local Ethics Committee, with informed consents obtained from all the patients.
Nucleic acid extraction: DNA was extracted from 200 μL of VTM using
spin-column based QIAamp Mini Kit (Quiagen, Hilden, Germany), according to the
Real-time PCR: The real-time quantitative PCR was performed with oligonucleotide
primer pairs and probe specific for the type-common region of HSV-1 and HSV-2
glycoprotein B (gB), as reported previously (Ryncarz et
al., 1999). The primers used were HSV-FP (5-TCC CGG TAC GAA GAC
CAG-3) and HSV-RP (5-AGC AGG CCG CTG TCC TTG-3) and the probe
was HSV-TCP (5-FAM-TGG TCC TCC AGC ATG GTG ATG TTG/C AGG TCG-TAMRA-3).
Amplification was carried out in an Applied Biosystem Sequence Detector 7500
machine, programmed for a four-step protocol: 2 min of incubation at 50°C
for AmpErase activation, 10 min at 95°C for polymerase activation and for
45 cycles: 15 sec at 94°C for denaturation, 60 sec at 58°C for annealing,
extension and data collection. Each 50 μL-PCR mixture contained 10 μL
of purified DNA, 840 nM concentrations of each primer and 100 nM probe in 1x
TaqMan universal PCR master mix (Applied Biosystems, Branchburg, New Jersey
USA). Negative controls were included in the extraction process between every
20 clinical samples. All the negative samples were tested twice (Ziyaeyan
et al., 2011).
Target amplicons were produced based on a standard PCR protocol by the above-mentioned
primers set and separated on agarose gel, then extracted and purified from gel
and cloned into the pTZ57R vector (Fermentas UAB, Vilnius, Lithuania) (Sambrook
and Russell, 2001). The plasmid containing target sequences was purified
using a commercial kit (Fermentas UAB, Vilnius, Lithuania) and its concentration
was determined spectrophotometrically. Quantification was carried out by drawing
standard curves using the serially diluted pTZ57R plasmid that contained the
target amplification product. Six positive control standards at 101,
102, 103, 104, 105 and 106
copies/reaction were used. The drawn standard curve was linear in 10 to 106
copies/reaction of the virus (R2 = 0.98). The copy numbers of clinical
samples were calculated automatically by applied biosystem sequence detection
software v 1.3.1.
Statistical analysis: Descriptive statistics and laboratory data were
analyzed using the SPSS statistical software package (version 15). Pearson correlation
test was used to detect the association between viral quantity and the age.
Fishers exact test was used to analyze the relationship between PCR results
and sex and t-test for that between PCR status and age.
From a total of 296 clinical specimens, one hundred and eleven (37.5%) corneal scraping samples tested positive for HSV DNA by real-time PCR. These positive patients consist of 69 males and 42 females. The copy number of HSV DNA, measured by the real-time PCR assay, ranged from 2.7x103 to 6.1x105 copies/500 μL of VTM, Mean±SD = 1.1x105±1.5x105.
However, as shown in Fig. 1, a relationship exists between
age and copy number .There was no relationship between gender and PCR status
(p = 0.581, Fishers exact test).
||Age distribution of 111 patient with positive real-time PCR
result and their HSV DNA copy numbers/500 μL viral transport medium
There were significant differences in age among patients with PCR positive
results and those with negative results (The mean ages were 48.4±17.1
years and 39.5±20.8 years among PCR positive and PCR negative patients,
respectively p = 0.0001, t-Test). No significant correlation between age and
load of HSV DNA was found (p = 0.079, Pearsons correlation test) (Fig.
Thanks to its efficacy, high accuracy and early detection, PCR has replaced
most previous conventional methods of HSV ocular infection diagnosis (Kakimaru-Hasegawa
et al., 2008). It seems that the results of quantitative analysis
of HSV DNA in herpetic ulcers, along with monitoring of HSV DNA levels could
be used to evaluate the efficacy of antiviral therapy. The patients enrolled
in the present study, were all with suspected HSK and referred to the clinics
for HSV DNA diagnosis in their eye lesions. Of the 296 cases, 111 (37.5%) were
found to be with HSV DNA. There have been very few reports on highly frequent
HSV asymptomatic shedding in ocular surfaces of healthy people with no corneal
HSV involvement, but many reports on shedding in saliva (Kaufman
et al., 2005). However, the use of well standardized PCRs can prevent
the effects of false positive results on the PCR results to a large extent (Leigh
et al., 2008). The fact that quantitative real-time-PCR can both
detect HSV DNA and evaluate the respective load can help follow the ACV treatment
course. Although resistance to ACV is reported in HSK (Choong
et al., 2010), the studies indicate that HSV resistant strains are
very limited in the region and in immunocompetent individuals (Ziyaeyan
et al., 2007b). Yet, some recent studies have demonstrated the possibility
of isolating resistant and sensitive strains from herpes-caused corneal lesions
at different intervals (Duan et al., 2009). Fortunately,
HSK is usually unilateral and is responsive to the conventional medications
with low toxicity (Hill and Clement, 2009; Tabbara
and Al Balushi, 2010).
The results of the present study indicate that HSV DNA diagnosis in corneal
ulcers is related to the patients age, i.e., the older the age of those
suspected with herpetic corneal ulcers, the greater the chance of HSV DNA diagnosis
in them. Although primary HSV infection in the region occur mainly during childhood,
the majority of HSK cases are due to the recurrence of latent infections which
aggravate the respective sufferers conditions at older age, as demonstrated
in the current study.
On the other hand, it was observed that the increased HSV copy numbers were
not significantly related to age in the patients with positive HSV DNA PCR.
However, as shown in Fig. 1, a relationship exists between
age and copy number. On the contrary, the HSV copy numbers in 6 patients aged
sequentially as follows, were significantly high; (1) male 34 years, HSV copies:
5.3x105; (2) male 29 years, HSV copies: 5.3x105; (3) male
27 years, HSV copies: 6.1x105; (4) male 24 years, HSV copies: 5.6x105;
(5) male 21 years, HSV copies: 1.1x105 and (6) female 6 years, HSV
copies: 6x105. Therefore, the relationship between the older age
and higher copy numbers in HSV patients is fading. We can then suggest that
the reason for the higher titer of HSV in these 6 patients might be initial
herpetic infection. High levels of HSV DNA in patients samples may indicate
active HSV reproduction in lesions resulting in corneal disease. Although the
damage to the cornea is not remarkable in cases with primary herpetic infections
in the eyes, it seems that the titer of the virus is high. In the present study,
the quantitative R-T PCR, used with fluorescent probe, possessed optimal sensitivity
and specificity and could enhance HSK diagnosis. As shown, the early and on
time ACV therapy can remarkably reduce the viral titer of the herpetic ulcers
(Deai et al., 2004) whcih is very helpful to
HSK treatment course. Our results suggest that HSK remains as an epidemiologically
important eye disease and improvement in the monitoring and detecting of ocular
herpetic disease by quantitative PCR method is informative and helpful to the
clinical diagnosis, decision for treatment and follow up.
The present study was supported by a research grant from the Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences. Our thanks are due to Hassan Khajehei, PhD for his linguistic copy editing.
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