Herpes simplex viruses (HSV) type 1 and type 2 belong to the Herpesviridae family and the Alphaherpesvirinae subfamily. The main characteristic of herpes simplex viruses is that they cause persistent latent (lifelong) infections. After the primary infection, which may or may not have clinical manifestations, a latent infection is established in the regional ganglia. Subsequent reactivations can also be with or without symptoms. Clinically, HSV infections can manifest themselves in different ways: gingivostomathitis, keratoconjunctivitis, encephalitis, genital infections and neonatal infections (1).
Most commonly, the HSV-1 infection appears in the orolabial region (labial herpes), and the HSV-2 infection in the anogenital region (genital herpes). New data, however, suggests a trend of growing prevalence of HSV-2 infections in the orolabial region and HSV-1 infections in the anogenital region. This can be explained with an increased frequency of orogenital contact (2).
Genital herpes is a sexually transmitted disease (STD) with a prevalence of 15% with or without symptoms, diagnosed using PCR in genital secretions of otherwise healthy women (3). The genital primo infection most often occurs in adolescents or young adults. The incubation period is usually from seven to ten days. The majority of infected subjects have no symptoms, while a small number develops vesicular or ulcerative lesions. In women these lesions can be found on the vulva, perineum, vagina and cervix. Lesions are usually followed by pain, inguinal adenopathy and dysuria. Symptomatic herpes on average lasts for about three weeks. After the primo infection, latency is established, usually in the lumbosacral ganglia. Reactivation can occur with symptoms (recrudescence) or without symptoms (recurrence). Lesions upon reactivation are usually limited to the genital region; the symptoms are less severe and last for a shorter period of time (4).
Asymptomatic viral shedding is an especial epidemiological problem in genital HSV transmission. Research indicates that HSV seropositivity is linked to viral shedding from the genital tract, even without a manifested infection. Between two reactivations (episodes of shedding) there is no evidence of viral presence in the genital tract (5,6). According to data taken from literature, viral DNA detected by PCR in genital secretions can be found in approximately 14% of immunocompetent subjects who do not present any symptoms of herpes (3).
HSV infections have a greater significance in HIV positive subjects and those who suffer from AIDS. HSV type 1 and 2 are among the most common causes of viral infections in these patients (7). Furthermore, studies show that HSV can influence sexual HIV transmission, and asymptomatic genital shedding is especially relevant in that aspect (8).
The aim of this study was to determine the frequency of asymptomatic shedding of Herpes simplex virus type 1 and 2 from the cervices of HIV positive women in the Serbian population, and note a possible correlation with the immunological status determined by the number of CD4+ T lymphocytes in these patients.
The study involved 30 HIV positive women from the Center for HIV/AIDS Institute for Infectious and Tropical Diseases Clinical Center of Serbia, aged nineteen to forty. All the subjects were informed about the nature of the research and were compliant. The subjects were divided into tree categories, depending on their CD4+ T cell count:
The subjects experienced no symptom of genital herpes, at the time the samples were taken nor in the past and received no antiviral therapy for herpes simplex virus.
The clinical samples were endocervical swabs taken from the subjects at the Center for HIV/AIDS and the Institute for Microbiology and Immunology and were processed at the Virusology Laboratory at the Institute for Microbiology and Immunology School of Medicine University of Belgrade.
Upon their acquirement, the swabs were dunked in 1ml of sterile saline and were kept at a temperature of -20˚C untill further processing.
The presence of herpes simplex virus in the endocervical swabs was determined using methods of molecular biology for determining HSV-1 DNA and HSV-2 DNA. The procedure involved: 1. extraction of DNA, 2. PCR (Polymerase Chain Reaction) for amplification of the desired DNA segment and 3. gel electrophoresis for visualization of the PCR product of a certain size.
Melted samples were centrifuged for 10 minutes at 3000 rpm. The sediment was used for DNA extraction using QIAamp DNA Mini Kit (QIAGEN INC., CA, USA). The extraction was done by manufacturer's protocol. The DNA extract was used for PCR.
The PCR reaction was done using primers for the timidin kinase gene (FW 5’-AGC GTC TTG TCA TTG GCG AA-3’ and REV 5’-TTT TCT GCT CCA GGC GGA CT-3’), for HSV-1 and primers for the DNA polymerase gene (FW 5’-CGT CCT GGA GTT TGA CAG CG-3’ and REV 5’-CAG CAG CGA GTC CTG CAC ACA A-3’) for HSV-2.
For the PCR reaction PCR Master Mix (Eppendorf, Hamburg, Germany) was used. The volume of the PCR mixture was 25 μl and it contribed of 20μl of PCR mix, and 5μl of the DNA extract. The program for PCR was: first step denaturation at 95˚C for 5 minutes, then a three step replication process (denaturation at 94˚C for 30 seconds, primer hibridisation at 58˚C for 30 seconds and extension of the DNA chain (elongation) at 72˚C for 1 minute). The replication process was repeated 40 times. After replication is completed, a final elongation step is done at 72˚C for 20 minutes, after wich the reaction if finalised at 4˚C.
Visualisation was achieved using gel electroforesis on a 1% agarose gel with etidium bromide (10mg/ml) and a 100 bp ladder. The desired PCR product was 342 bp in size for HSV-1, and 445 bp in size for HSV-2. (Fig. 1)
Of the 30 samples examined, HSV DNA was detected in genital secretions of 11 HIV positive women (36,7%). HSV-1 was detected in one (9,1%), and HSV-2 was detected in eight patients (72,7%). Two of the HIV positive women were positive for both types of HSV (18,2%).
Total frequency of asymptomatic shedding of HSV-2 in genital secretions of HIV positive women was greater then that of HSV-1 (33,3% vs. 10%). (Table 1)
The smallest percent of positive samples (12,5%, 1/8) was detected in the second category of subjects (from 200 to 499 CD4+ T cells per mm3), all of witch was HSV-2. In the first category of HIV positive women (500 or more CD4+ T cells per mm3) 3 out of 6 (50%) were positive for HSV-2 and none were positive for HSV-1. In the third category of HIV positive women (less than 200 CD4+ T cells per mm3) 7 out of 16 were positive for HSV (43,75%), of which 1 was positive for HSV-1 (6,26%), 4 were positive for HSV-2 (25%) and 2 were positive for both types of HSV (12,5%). (Table 2)
A number of epidemiologic and clinical studies pointed out the influence of genital HSV infection on the course and transmission of HIV infection, and asymptomatic shedding is especially important in this aspect (9,10,11).
Recent studies show an increase of prevalence in asymptomatic cervical shedding in HIV positive women in comparison to HIV negative women. In a study conducted on HSV-2 seropositive women, PCR showed an increase in asymptomatic HSV-2 genital shedding in HIV positive women (43%) relative to HIV negative women (22%). In this study a prevalence of asymptomatic genital HSV shedding of HIV positive women was found to be 36,7%. Results also indicate a higher frequency of HSV-2 asymptomatic shedding relative to HSV-1 (33,3% vs. 10%). These findings are expected, with HSV type 2 usually causing genital herpes (1). An asymptomatic shedding of both HSV types was shown in two HIV positive women. This finding is in accordance with earlier results of different studies showing that antibodies specific to one type of HSV are not protective for the other HSV type (6).
These results raise a question of which factors influence asymptomatic HSV shedding on the cervix of HIV positive women.
In different studies, a correlation was shown between quantities of HIV1-RNA and HSV-DNA in genital secretions of HIV positive women with an asymptomatic HSV infection. Research indicates that HSV shedding is up to two times greater in HIV patients than in HIV negative ones. It is generally supposed that this increase in HSV genital shedding is a consequence of HIV-induced immunosuppression or an effect of some other systemic or local cofactors on HSV (5,9). For example, studies show that cervical HSV shedding is affected by a CD4+ T cell number lower than 200/mm³ of blood, the use of contraceptive medication and serum deficiency of vitamin A (12). In this study no correlation was found between CD4+ T cell count and asymptomatic HSV genital shedding of HIV positive women, because the lowest percentage of positive HSV findings was proven in the second category of HIV positive women (12,5%), in which CD4+ T cell count was 200-499/mm³ of blood. Still, presence of both viruses in the genital tract was proven only in patients belonging to the third category, where CD4+ T cell count was lower than 200/mm³.
In addition, one of the most important factors affecting the severity of clinical manifestations and the frequency of HSV reactivation in HIV positive individuals is the specific CD8+ cytotoxic T cell response (13). Since deterioration of CD8+ cytotoxic T cell function correlates with the progression of the disease, it is obvious that clinical manifestations of HSV infection will be more frequent.
On the other hand, it is considered that HSV and HIV can interact on the molecular or cellular level, which can affect the intensity of HIV replication and the course and facilitation of transmission of HIV infection (9,10,11). In some in vitro experiments it was shown that HSV can stimulate HIV replication with its early genes (ICP 0, ICP 4, ICP 27). This stimulation occurs by activation of HIV-LTR transcription and represents the so-called cofactor transactivation (14). This assumes that HSV presence in the cervical epithelium increases HIV shedding and with it the risk of sexual transmission (9,15). Furthermore, HSV could facilitate the acquiring of HIV infection by disruption of the epithelial barrier and mobilization of CD4+ T cells in the zone of infection (16).
The results of this study show a significant frequency of asymptomatic HSV shedding in genital secretions of HIV positive women, whereas HSV-2 is more often found than HSV-1. Furthermore, an asymptomatic presence of both viruses was shown in genital secretions of two women. Regarding the immunological status of HIV positive women, no correlation was found between the asymptomatic HSV shedding and CD4+ T cell count.
These results show the medical and the epidemiological importance of asymptomatic genital HSV infection for both herpes transmission and HIV infection pathogenesis. In that aspect, further research is necessary that will definitely establish the importance of preventive measures, early discovery and treatment of genital herpes in controlling the HIV infection.
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