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APPLICATION OF DIRECT IMMUNOFLUORESCENCE TEHNIQUE IN DETECTION OF RECURRENT GENITAL HERPES

Vesna Miloevi1, Gordana Kovaevi1, Ivana Hrnjakovi-Cvjetkovi1, Vera JerantPati1, Jelena Radovanov1, Gorana osi 2 , Marina ermanov3 1 Institute of Public Health of Vojvodina, Center of Virusology , Serbia 2 I nstitute of Public Health of Vojvodina , Center for control and prevention disease, Serbia 3 University of Novi Sad, Medical Faculty, Serbia

Adresa autora: Prof. dr Vesna Miloevi, Institut za javno zdravlje Vojvodine, 21000 Novi Sad, Futoki put 121, e-mail : virusologija@izjzv. org. rs.

Summary

Genital herpes infections are extremly common worldwide, and herpes simplex virus type 2 is the major cause of genital herpes. Although the majority of genital herpes is due to HSV2, an increasing rate is recognized due to HSV1 becauseof changed in sexual behaviour. The study took place at the laboratory of the Centre for virology, Institute of Public Health of Vojvodina. During the period of 10 years, 814 patients were examined, 580 males and 234 females. The specimen for the direct immunofluorescence test (DFA) was urethral smear taken from males and endocervical smear taken from females. DFA confirmed the virus infection in 30% ( HSV1 in 8,1 %, and HSV2 in 20,8%) of 580 specimen taken from males. DFA confirmed the virus in 22% (HSV1 in 4,2% and HSV2 in 17,5%) in female group. Among of both sexes, infection with HSV1 was more frequent in age group 20-29 years. Females aged from 20-39 and males aged from 30-39 were the most exposed to infection caused by HSV2. In the first year after initial infection, was found a statistically significant reduction in the number of episodes of genital herpes caused by HSV1, while reducing recurent genital herpes caused by HSV2 wasn't statistically significant. Conducted studies have confirmed the benefits of laboratory diagnosis of genital herpes. DIF technique is rapid, specific and applicable in diagnosis of recurent genital herpes . Key words: direct immunofluorescent technique, herpes simplex, recurrent genital herpes

INTRODUCTION The family of Herpesviridae includes almost hundred different types of viruses. Only eight of them can cause the infection in human population. They are divided into subfamilies, Alpha, Beta and Gammaherpesvirinae by the specificity for the host, the period of reproductive cycle and the latency period characteristics. The herpes simplex viruses type 1 and 2, subfamily Alphaherpesvirinae, can cause persistent infection with recurrent symptomatic and asymptomatic reactivation (1,2). Genital herpes is very common sexually transmitted disease, caused by the infection of herpes simplex virus type 2 in most cases. However, the change of sexual habits makes herpes simplex type 1 more often the cause of the first or recurrent genital herpes (3,4,5). People with solid immune system infected with HSV can have frequent, painful and recurrent genital lesions, followed by different psychological problems. Although the symptoms of acute first episode of the genital herpes caused by HSV1 or HSV2 are mostly the same, recurrent infections are less frequent and less intense in HSV1 infections (5). The severity and clinical manifestations of the first episode and reactivating infections of HSV2 are less intense for those who already have HSV1 infection. The prevalence of the HSV1 infection is larger than HSV2 infection in most

parts of the world. In general population, approximately 20-60% sexually active men and women have antibodies for HSV2, and 40-90% of the population have antibodies for HSV1 (4). People with HSV2 infection are in three times bigger risk for acquiring HIV infection, and those with both HIV and HSV2 infection transmit the HIV infection more often onto their sexual partners (3,6). The variety of genital herpes clinical manifestations, with frequent atypical symptoms, overlap with other infective or non infective causes of genital ulcers and asymptomatic infection which remain undiagnosed, emphasizes the importance of laboratory diagnosing of HSV infections. The most recent laboratory methods in diagnostics of genital HSV infection include: cytological smears of visible lesions, serology methods, HSV antigen immunofluorescence or immunoenzyme proving, DNA hybridization technique, Real Time-PCR and isolation of the virus in culture (7). Previous experiences with these methods showed that sensitivity of laboratory methods depends on the type of the lesion and also occurrence of the episode (5,7,8). Aim: The aim of the paper is to determine the rate of the urogenital infection due to herpes simplex viruses depending on the type of the virus, sex, and age of the patients in Novi Sad and the surroudings.

MATERIAL AND METHODS

Retrospective study had covered the period of 10 years (01.01 2000 01.07. 2010). Data were used from the Protocol of virusological analyses, Center of Virusology, Institute of Public Health of Vojvodina. All samples were taken from patients with clinical indications and recommended by urologists and gynecologists for further virusological HSV diagnostics. At the moment of check-up patients had various urological and genital difficulties or visible lesions. As a specimen for direct immunofluorescent technique (DIF) urethra smear scarification is performed at men with suspected acute recurrent herpes or those changes were suspected to HSV infection. Endo-cervical and vaginal smear was taken from women who had visible lesions or from those who had continual difficulties with suspected HSV infection. Results were obtained by DIF testthe Micro Track HSV/HSV2 Direct Specimen Identification/Typing Test (Trinity, Biotech Ireland) for herpes simplex virus diagnostics in vivo from cutaneous lesions. Test is conducted in a way that on the original plates is placed urethral smear taken from males or endo-cervical smear taken from females. Smears are dried at the room temperature and fixed in acetone for 10 minutes. Slides that are not treated by fluorescent antibodies immediately are kept on

the temperature of 700C. Fixed preparations are treated with 30l conjugates and incubation in the moist chamber is performed for 15 minutes at the temperature of 370C or 30 minutes at the room temperature. After performed incubation slides are rinsed 10 to 15 seconds in distilled or de-ionized water. A drop of Mounting medium is added to dried preparations and slides are examined under immuno-fluorescent microscope with lens having the enlargement capacity of 40 times. Cells infected by certain virus type showed fluorescent green spots, typical for HSV1 or HSV2 infection. Positive result indicates presence of at least one cell that completely fluoresces or visible immuno-fluorescent inclusions in nucleus. Characteristic fluorescent color was not registered in negative preparation. Statistical analysis The collected data were stored in a cpecialy created database on personal computer. Statistical processing was done under aplicaton software Numerical data are presented as SD, as median number (%). Statistical significance was determined via 2 test with a significance level of p>0,05 and McNemar test with a significance level of p< 0,100.

RESULTS The sample included 814 patients, 580 males (71%) and 234 females (29%) whose genitourethral smears were tested for HSV infection using DIF test ( figure 1). Average age of examinee was 35,6 years for men and 34, 3 years for women. Out of 580 men were examined, in 173 (30%) cases the HSV infection was proven by DIF test, 52 (8,9%) had HSV1, and 121 (20,8%) had HSV2 infection (figure 2). 2 test showed that there is a statistically significant difference between the frequency of HSV types in proven infections (p< 0,05), and also that HSV2 is significantly more frequent cause of urogenital infections in male group. Out of 234 women whose vaginal or endocervical smear were examined by DIF test, in 51(22%) cases HSV infection was proven. HSV1 was diagnosed in 10 (4,2%) cases, and in 41 (17,5%) was diagnosed HSV2 infection (figure 3). 2 test showed that HSV2 was statistically significant more frequent in genital infections in female group (p< 0,05) than HSV1. Representation of HSV types according to sex is given in figure 4. There is statistically significant difference in frequency of the HSV1 as cause of infection in favour of male group p <0,05 rather than in female group. There is no statistically

significant difference in the frequency of HSV2 as a cause of infection between sexes, p> 0,05. In male group, urogenital HSV infection is the most frequent in the age group 20-39 years. HSV1 infection is the most frequent in the age group 20-29 years, but HSV2 infection is the most frequent between 30 and 39 years. In female group, the most of HSV1 infection was in the age group 20-29 years, but HSV2 infection most frequently appeared in the age group 20 to 39 (table 1). 50 patients were monitored for HSV more than 6 months to assess of renewed appearance of genital lesions. With McNemar test it is established that there is statistically significant reduction of the number of recurrent herpes episodes caused by HSV1 rather than those caused by HSV2, p=0,0044. The reduction of recurrent herpes caused by HSV2 had no statistical significance, p=0,6625 (table 2). In 29 patients, during the first year after appearance of the infection, were registered from 1 to 5 episodes of recurrent herpes caused by HSV2, and in 10 patients with HSV1 infection from 1 to 3 episodes. In five patients who had the proof of the infection with both HSV1 and HSV2 types by DIF test, were registered from 1 to 3 episodes of recurrent herpes. Cmpared to HSV1, HSV2 is more often diagnosed both in vaginal and endocervical smear in the group of 86 women who have tested both of these smears by DIF test (table 3). DISCUSSION In this paper results of urogenital smears obtained by method of direct immmunofluorescence (DIF) are analyzed. In total, 814 patients are examined, 580 (71%) men and 234 (29%) women. Patients had suspected recurrent genital HSV infection. 30% of male group and 22% of female group were diagnosed with HSV infection by DIF method. Statistical analysis showed no statistically significant difference between the number of infected women and men. This fact doesnt match the information in other studies, which show that there is always more infected women than men in sex structure. (3, 4, 5, 9). The results in this essay corresponded to the research of Italian author Causini et. al. that demonstrate equal representation of genital herpes between sexes (9). The authors of the study explain the results by the age of women who were younger than men, so that they were exposed to possible infection shorter period of time, as well as they could have less sexual partners (9). Similar results presented by Spanish authors, emphasize that seroprevalence of genital infection is equally present between men and women in Spanish population (10). Age similarity of examined patients affected the results in this study, as well as possibility that the infection was apparent in the male group so that they have been advised more often by urologist to do the test for HSV infection, and it is possible that in female group infection is not symptomatic and was unnoticed. Also the results

affected methods which were used to detect the viral infection. Every method has different grade of selectivity, sensitivity, utility and cost . For detection of HSV the golden standard is cell culture, however, it takes few days to get the results using this method (2, 8). With DIF test the results are obtained in few hours, so in this study is emphasized the utility of laboratory diagnostics of genital herpes using this method, which is specific and applicable in diagnostics of recurrent genital herpes. But, sensitivity of the method depends on the stage of lesion and also wether the examined population has first or recurrent episode of genital herpes. Patients who took part in this study had the suspect recurrent genital HSV infection and diagnostics with type specific monoclonal antibodies depended on clinical manifestation of the lesion in genital track and its duration. Besides the detection of HSV antigen by DIF test is in lower correlation with infectiousness of the virus, so that false negative results do not recognize patients who excrete the virus. The results in this study, obtained by DIF test, showed that genital herpes is statistically significantly more frequent caused by HSV2 within infected patients of both sex. Although, the virus is more often diagnosed in female group (80, 4%) compared to males (69, 9%), this difference of the frequency of HSV2 infection according to sex is not statistically significant. In the study, 19, 6% of infected women and 30, 1% infected men are diagnosed with HSV1 infection as cause of genital herpes. Though the term, genital herpes, the most often is used as a synonym for HSV2 infection, also HSV1 can cause infection of urogenital region. There are information that today, genital herpes more frequently is caused by HSV1 than before, caused by sexual habits (2-8). In this paper results of DIF test show that HSV1 is statistically significant more frequently cause of genital infection in male group p< 0,05 compared to female group. This facts can be explained by sexual habits of tested population (3, 4, 11). The most of the infected patients are in age group 20-39 years. In male group the number of infected men is increasing after the age of 24, but in female group the number of infected patients has not change between the age groups. In past decades increased number of genital herpes caused by HSV1, especially among young population is noticed (2-7). The most of the infected by HSV1, men as well as women examinee were in the age group between 20 and 29. The HSV2 infection overcame in the age between 30 and 39. As well as other STDs, promiscuous behaviour is a risk factor for spreading genital herpes. The prevalence of HSV genital infections is increasing with age and number of sexual partners. The percentage of infected persons is higher in certain ethnic groups, as well as in populations with lower socioeconomic and cultural status (9). Clinical course of the first episode of genital herpes, caused by herpes simplex virus type 1 or 2 is very similar (5, 9). Repetition of recidive genital herpes, with apparent signs or without symptoms can be very often in first one or two years of getting the infection. However, frequency and severity of recidives is less in HSV1 infections compared to HSV2 infection (9,10). Toward the evaluation of the frequency of acute episodes of recurrent herpes, during 6 months period and more, the follow-up has been done for 50 male examinees. Repetitive testing during separate episodes of genital lesions and DIF testing can provide move laboratory conformation of genital herpes. It is established statistically significant difference in reduction of the rate of recurrent

herpes caused by HSV1. But, in HSV2 infection there was no decrease of the number of recidives during the first year after getting the infection. The number of episodes of recurrent genital herpes, caused by HSV2 , was detected in 48% observed examinee and it was between 1 and 4. Recurrent herpes, caused by HSV1 was found in 30%, and the number of repetition was between 1 and 3. Five patients had the infection caused by both HSV1 and HSV2, and the number of repetition was between 1 and 3 episodes of recurrent herpes. The obtained results are in accordance with research of other authors. Gupta et al., presented the fact that in the absence of antiviral therapy, average number of recidive HSV2 infection was around 4 in the first year after the prime infection (3). Benedetti et al., noticed that average number of relapsing HSV infection was between 1 and 5, In their study, 70 90% patients had recurrent herpes, caused by HSV2 in the first year after the infection, while 20 50% of patients had recurrent herpes caused by HSV1. Same authors came to the conclusion that there was correlation between initial infection and later rate of relapses. Relapses of genital herpes, appeared two times more often with patients who had more severe and prolonged primary episode of the infection (12). Clinical diagnosis should be confirmed by laboratory tests, including serotyping (5, 13, 14). Diagnostics type specific monoclonal antibodies, tagged with fluoresceine, which was used to detect viral HSV antigen in this paper is specific, quick, simple and cheap method (13,14). The possibility to determine serotypes of HSV has not only diagnostic but also prognostic significance in determination of antiviral therapy. The detection of antigen with DIF method depends on clinical stage of the lesion in genital tract, as well as if it was the first or recurrent episode of genital herpes (5, 8, 14). The DIF technique is sensitive if smear is taken just after the appearance of the lesion in genital tract. In the case of 9 female patients who were followed-up for recurrent herpes, in the first testing with DIF method, HSV2 antigens were negative, but in second one they were positive. When there is a high index of clinical doubt for genital herpes, toward the conformation the diagnosis, it is advised to repeat the examination with DIF method (8). For 86 female patients it was done of both vaginal and endocervical smear in the same time, DIF HSV diagnostics, because they had unclear symptoms for HSV infection. CONCLUSION This study confirms the benefit of laboratory diagnostics for genital herpes. DIF technique is specific and applicable in diagnostics of recurrent herpes. However, sensitivity of this method depends on the quality of the obtained sample and the experience of the person working on the microscope. Repeated samples of separate episodes of genital herpes and DIF testing can provide safer laboratory conformation of genital herpes.

REFERENCE:

1.Jerant-Patic V. Medicinska virusologija. Beograd: Zavod za udzbenike i nastavna sredstva; 1995. 2. Chayavichitsilp PB, Joseph V, Krakowski, Andrew C, Friedlander Sheila F. Herpes Simplex. Pediatrics in Review 2009; 30: 119-130. 3. Gupta R, Warren T, Wald A. Genital herpes. The Lancet 2007; 370(9605): 2127 2137. 4. Mikloka Z, Brki L, Cvija, H, Maak-afranko , Zeni L. Imunopatogeneza infekcije herpes virusom u ovjeka: implikacije za razvoj cjepiva. Struni rad. Medix 2005; 11 (11): 78 81. 5. Singh A, Preiksaitis J, Ferenczy A, Romanowski B. The laboratory diagnosis of herpes simplex virus infections. Can J Infect Dis 2005; 16(2): 9298. 6. Ashley RL. Laboratory techniques in the diagnosis of herpes simplex infection. Genitourin Med 1993; 69:174-183. 7. Rigopoulos D, Malouchou K, Alevizos A, Larios G, Papadogiorgaki H, Lima K, Antoniou C. Extensive Atypical Genital Herpes Simplex Type 2 Infection as an Initial Manifestation of Acquired Immune Deficiency Syndrome. Acta Dermatovenerol Croat 2008; 16: 3. 145-148 8. Lafferty WE, Krofft S, Remington M, R Giddings, C Winter, Cent A, et al. Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a high-prevalence population. J Clin Microbiol 1987; 25 (2): 323-326. 9. Cusini, M, Cusan M , Parolin K,Scioccati L. Seroprevalence of herpes simplex virus type 2 infection among attendees of a sexually transmitted disease clinic in Italy. Sexually Transmitted Diseases 2000 ; 27 (5): 292-295. 10. Corbeira PG, Dal Lorenzo RA, Granizo JJ, Garca-de-Lomas J .Is sexual transmission an important pattern for herpes simplex type 2 virus seroconversion in the Spanish general population? Journal of Medical Virology 1999; 59(2): 194197. 11. Jonsson MK, Wahren B. Sexually transmitted herpes simplex viruses. Scandinavian Journal of Infectious Diseases, 1651-1980 2004; 36(2): 93 101. 12. Benedetti J, Corey L, Ashley , Recurrence Rates in Genital Herpes after Symptomatic First-Episode Infection. Ann Intern Med 1994;121:847-854. 13. Sen P, Barton SE. Genital herpes and its management. Clinical Review. MJ 2007; 334:1048-1052 .

14. Miloevi V, Jerant-Pati V, Hrnjakovi-Cvjetkovi I, Kovaevi G, Radovanov J, ermanov M. Dijagnoza genitalnog herpesa direktnom imunofluorescentnom tehnikom. VII kongres mikrobiologa Srbije. Mikromed 2010, Beograd 2010.

Abbreviations: HSV- herpes simplex virus DIF direct immunofluorescent technique

29%

males females
71%

Figure 1. Distribution sexes in the sample

20,8%

8,9%

without infection HSV1 HSV2


70,3%

Figure 2. The prevalence of infection in concerning the type of virus in males

17,5% 4,2%

without infection HSV1 HSV2


78,5%

Figure 3.

The prevalence of infection in concerning the type of virus in females

69,9%

80,4%

30,1%
19,6%

HSV1 HSV2

Figure 4. Representation of HSV types in HSV positive patients

males

females

Table 1. Disribution of HSV infection by age groups Age distributio n < 19 20-29 30-39 40-49 <50 MALES MX 17,9 24,9 33,9 44,3 58,1 HSV1 2 18 16 7 9 HSV2 1 36 45 17 20 ZX 17,1 25,2 33,7 44,0 58,3 34,3 0 6 2 1 1 10 FEMALES HSV1 1 16 15 4 5 41 HSV2

35,6 52 121 Total Legend: M X average age in male group Z X average age in female group

Table 2. McNemar test for HSV1 and HSV2 in patients with performed DIF monitoring DIF test for HSV1 Monitoring I find McNemar test positive negative positive 4 10 I control negative 0 11 McNemar test positive negative I find positive 11 12 I control negative 9 7 DIF test for HSV2

Table 3. Female patients that tested both vaginal and endocervical smear in the same time for HSV infection DIF Sample Poz n Vaginal swab % n endocervical swab % 3,4% 36% 59,6% 16,2% 79,2% 4,6% Legend: * without performed diagnostic 2,3% 3 37,2% 31 60% 51 9,3% 14 86,1% 68 4,6% 4 86 2 HSV1 neg 32 * 52 poz 8 HSV2 neg 74 * 4 86 Total