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Prevalence of Herpes Simplex Virus Type 2 Antibody in Cameroon

EIS-HÜBINGER, ANNA MARIA DVM*; NYANKIYE, EMMANUEL MD; BITOUNGUI, DIDIER MBOUA MD; NDJOMOU, JEAN MSc

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Background Genital herpes is one of the most common sexually transmitted diseases. As a leading cause of genital ulceration, herpes genitalis plays a role in facilitating the transmission of HIV. Although HIV infection is most prevalent in Cameroon, information is lacking about prevalence of herpes simplex virus (HSV) type 2 infection in this country.

Goal The goal was to determine the prevalence of HSV-2-specific antibody in blood specimens from individuals in Cameroon.

Study Design Blood specimens were randomly collected from 410 clinic attendees (215 males, 195 females) in Douala, the most populous city in Cameroon. One hundred fifteen of the individuals (28.0%) were HIV-infected. Samples were tested by a type-common HSV IgG enzyme immunoassay not discriminating between HSV-1 and HSV-2 antibodies and by two glycoprotein G-2-based enzyme immunoassays for detection of HSV-2-specific antibody.

Results All but three blood samples were positive for type-common HSV IgG antibodies. Sixty-seven specimens (16.3%) were concordantly negative for HSV-2 antibody by both assays, and 287 (70.0%) specimens were concordantly positive. Fifty-six specimens (13.7%) yielded discrepant results between the two assays.

Conclusion On the basis of specimens with concordantly positive results, the overall HSV-2 seroprevalence was 70.0%. HSV-2 seroprevalence was significantly higher among HIV-infected individuals than among HIV-negative ones. Because of the serious morbidity and mortality caused by HSV-2, effective programs are needed to halt the spread of HSV-2 infection in Cameroon.

Blood specimens from clinic attendees in Douala, Cameroon, were tested for herpes simplex virus type 2-specific antibody. Overall seroprevalence was 70.0%. Rates were higher in HIV-infected individuals than in HIV-negative ones.

From the *Institute of Medical Microbiology and Immunology, University of Bonn, Germany; the †Chest Clinic and ‡Sexually Transmitted Disease Clinic, Douala, Cameroon; and §University Center Hospital, Yaounde, Cameroon

Reprint requests: Dr. Anna Maria Eis-Hübinger, Institute of Medical Microbiology and Immunology, University of Bonn, Sigmund-Freud-Str. 25 D-53105, Bonn, Germany. E-mail: Anna-Maria.Eis-Huebinger@ukb.uni-bonn.de

Received for publication September 10, 2001,

revised January 8, 2002, and accepted January 10, 2002.

GENITAL HERPES is one of the most common sexually transmitted diseases (STDs) in humans. 1,2 Infection is mainly caused by herpes simplex virus type 2 (HSV-2) and less commonly by HSV type 1 (HSV-1). 3,4 The infection can be associated with serious morbidity, such as painful vesiculo-ulcerative disease of the genitals and adjacent areas and inguinal adenopathy. Primary infection can be complicated by fever, aseptic meningitis, sacral radiculomyelopathy, and urinary retention. 5,6 In addition, patients often also have life-long psychological stress as a result of the unpredictable genital herpes recurrences and the risk of transmitting viral infection to sexual partners. Another major problem associated with genital herpes in females is the risk of neonatal herpes acquired by contact of newborns with infected genital secretions during delivery. 7,8 Furthermore, herpes-associated genital ulcerations play a role in facilitating sexual transmission of HIV. 9

Unrecognized infections and asymptomatic viral shedding appear to be the key factors in transmission of infection. 10 In a recent study in the United States, HSV was detected in genital secretions by viral culture on 3.8% of days on which culture specimens were obtained from individuals who had antibody to HSV-2 but who reported having no history of genital herpes. 11 With use of the sensitive polymerase chain reaction, HSV-2 shedding was found on average 28% of the days on which samples were obtained from healthy, immunocompetent women. 12 Thus, identification of HSV-2-infected individuals highly relies on antibody identification.

Type-specific discrimination of the HSV antibodies, however, was hampered for a long time by serological cross-reactivity caused by extensive antigenic homology between HSV-1 and HSV-2. 13,14 Until recently, type-specific serology was restricted to a few specialized laboratories performing in-house-generated Western blotting, which is still considered the “gold standard” for diagnosis. 15,16 However, this procedure is time-consuming and too cumbersome to be widely applied to large-scale studies. Recently, new enzyme immunoassays (EIAs) based on preparations of glycoprotein G from HSV-2 (gG-2) or HSV-1 (gG-1) have become available for detection of HSV type–specific antibodies. 17 Due to the extensive differences between gG-2 and gG-1 in size, sequence, and structure, serological cross-reactivity is very limited. 18,19

Since information about HSV-2 seroprevalence in Cameroon is lacking, we examined the HSV-2 seroprevalence by two HSV gG-2–based immunoassays in blood samples from 410 adults attending 3 hospitals in Douala, the most populous city in Cameroon.

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Methods

Study Population

A total of 410 EDTA blood samples were collected from randomly selected adults (215 males, 195 females) in 1998 in three different hospitals in Douala. One hundred eighty-eight specimens were obtained from individuals (110 men, 78 women) attending a pulmonary clinic (group I), 161 specimens from individuals (79 men, 82 women) attending an STD clinic (group II), and 61 specimens from individuals (26 men, 35 females) attending a general hospital with symptoms suggestive of HIV infection (group III). The minimum age of the individuals was 20 years. In group I, the mean age was 34 years (range, 20–76 years); in group II, 28 years (20–79); and in group III, 33 years (20–50). One hundred fifteen (63 males, 52 females) of the 410 persons (28.0%) were seropositive for HIV in testing with Enzygnost Anti-HIV-1/2 Plus (Dade Behring) and Wellcozyme HIV Recombinant (Murex Diagnostics). In group I, 64 persons were HIV-seropositive (34.0%; 39 [35.5% of] males, 25 [32.1% of] females); in group II, 28 were HIV-seropositive (17.4%; 14 [17.7% of] men, 14 [17.1% of] women); and in group III, 23 were HIV-seropositive (37.7%; 10 [38.5% of] men, 13 [37.1% of] women).

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Laboratory Methods

All plasma samples were tested by a type-common HSV IgG EIA and two HSV-2–specific EIAs. The type-common assay (Enzygnost Anti-HSV/IgG, Dade Behring), which does not discriminate between HSV-1 and HSV-2 antibodies, uses as antigen HSV-1 strain McIntyre, harvested from the supernatant of infected permanent simian kidney cells. The two EIAs for detection of HSV-2-specific antibody are based on HSV-2 gG-2. Assay A (ETI-HSVK-G 2, Sorin Diagnostics Biomedica) is based on recombinant gG-2 produced by the baculovirus system, and assay B (Cobas Core HSV-2 IgG EIA, Roche) is based on gG-2 of HSV-2 grown in tissue culture and purified by lectin affinity procedure. All assays were carried out according to the instructions of the manufacturers. Assay A was performed manually and assay B was processed automatically by the Cobas Core II Immunoanalyzer (Roche). Results were interpreted in accordance with the manufacturer's instructions. To obtain comparable results, antibody reactivity was expressed as s/co values (optical density of the sample/optical density of the cut-off).

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Statistical Methods

Data were analyzed by chi-square test.

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Results

The results of testing for HSV type–common and HSV type 2–specific antibodies are summarized in Table 1. Overall, 99.3% (407/410) of the individuals tested seropositive for HSV. Only a 20-year-old female and a 39-year-old man attending the STD clinic and a 38-year-old man attending the pulmonary clinic tested seronegative for HSV.

TABLE 1

TABLE 1

Testing by two EIAs on the basis of HSV gG-2 yielded the following results: 287 blood specimens (70.0%) tested concordantly positive for HSV-2–specific antibody, and 67 (16.3%) tested concordantly negative for HSV-2–specific antibody; the three blood specimens that tested negative in the type-common HSV assay were included in this number.

Altogether, 56 specimens (13.7%) yielded discrepant results. Eleven blood samples (2.7%) were positive in assay A, and were equivocal in assay B. In most cases (36 samples) assay A yielded a positive result while assay B was negative. However, among these specimens, 13 that had to be classified as negative by assay B showed reproducible reactivities close (s/co ≥0.8) to that of the cut-off value. An additional nine specimens repeatedly produced reactivities at an elevated value (s/co > 0.75 < 0.8).

On the basis of specimens with concordantly positive results, the HSV-2 overall seroprevalence was 70.0% (287/410). There were no significant differences between the three individual groups (persons attending the pulmonary clinic: 136/188 = 72.3%; persons attending the STD clinic: 108/161 = 67.1%; persons attending the general hospital: 43/61 = 70.5%).

Overall, males and females had similar seroprevalence rates (147/215 = 68.4%, versus 140/195 = 71.8%). Only in the youngest female age group was seropositivity higher than in the age-matched male group. The difference was not significant, however. The data on age-related HSV-2 seroprevalence are shown in detail in Table 2.

TABLE 2

TABLE 2

HSV-2 seroprevalence was significantly higher in HIV-infected individuals than in HIV-negative individuals (98/115 = 85.2%, versus 189/295 = 64.1%;P < 0.001). The difference in HSV-2 prevalence between HIV-infected and -noninfected individuals was significant in both males (54/63 = 85.7%, versus 93/152 = 61.2%;P < 0.01) and females (44/52 = 84.6%, versus 96/143 = 67.1%;P < 0.05). Seven (6.1%) of the HIV-infected individuals were concordantly HSV-2-seronegative; the samples from 10 individuals (8.7%) yielded discrepant results. There was no difference in HSV-2 seroprevalence among the three different groups of clinic attendees with HIV infection (group I, 85.9%; group II, 82.1%; group III, 86.9%).

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Discussion

The aim of this study was to provide information on HSV-2 seroprevalence in Cameroon. For that purpose, newly developed tests using the type-specific glycoprotein gG-2 as antigen were applied. Although not completely free from some inherent shortcomings, 17 these assays are suited for large-scale studies, especially in high-prevalence populations. 20 The two EIAs that were used in this study have been extensively evaluated. 21–23 Both assays are commercially available and can be applied to serum or plasma. Assay A is performed in a microtiter plate format with 8-well strips, requiring standard pipetting equipment, facilities for incubation in a humid chamber at 37°C, and a 96-well plate reader that measures absorbance at 405, 450, and 630 nm. Assay B is a fully automated EIA without predilution of the specimen. Because the assay is based on antigen-coated beads, use of the appropriate autoanalyzer (Cobas Core) is required. The costs (without labor) to perform assay A are in the range of Euro 3.5 and in the range of Euro 9 for assay B, the latter including a calculation of the costs for the autoanalyzer.

The current study shows that HSV-2 is a common pathogen among the studied populations in Cameroon (overall seroprevalence, 70.0%). The seroprevalence increased with age, whereby seroprevalence was slightly higher in young females than in young males. The more rapid increase in HSV-2 antibody in females might be due to a higher risk of viral transmission with sexual exposure or an earlier initiation of sexual activity. To a certain degree, the high proportion of indeterminate results in the youngest age group that declines in the older age groups might be because the kinetics of detectable seroconversion to gG-2 positivity take a longer time 17 and low-level antibodies might be measured in some specimens. In the age group of 26 to 30 years, HSV-2 seropositivity was more than 70%, indicating that infection is predominantly contracted in young adulthood.

The gender difference in HSV-2 seroprevalence in young adults in the current study is in agreement with results from other countries, including those from tropical Africa. 24–29 However, in the current study, there was no difference in seroprevalence between men and women older than the age of 25 years. Comparison with other reports is limited by the small number of studies carried out in tropical Africa that examined males and females in the same study population. Furthermore, seroprevalence data from individuals of the older age groups are limited. In a study carried out in a rural Ugandan population of mainly peasant farmers with an HIV infection rate of about 5%, HSV-2 seroprevalence in the oldest age group (≥45 years) was more divergent (76.5% for women, 57.6% for men) 28 than in the current study. Similar results were obtained in a study investigating specimens from randomly selected individuals in rural Tanzanian communities 29 (seroprevalence of 79% [22/28] for women aged 35 to 54 years and 64% [14/22] for men; data for HIV infection not given).

In contrast, in a study of a population that was more similar to the population we investigated, i.e., attendees of an urban Tanzanian STD outpatient clinic of whom 25.3% were HIV-infected, no male–female differences in HSV-2 seroprevalence were found beyond the age of 30 years. 30 Thus, at least for the population of STD clinic attendees in our study, similar results have been obtained in other investigations. Hence, the results might indicate that in tropical Africa the gender-specific HSV-2 seroprevalence rate might differ in a population-specific manner from patterns commonly seen in other parts of the world.

The most probable reason for the relatively high seroprevalence in the studied male populations of Douala, Cameroon, could be men's sexual activity and behavior. In particular, unmarried young men are often sexually active with multiple partners. Moreover, especially among men, genital herpes is considered of minor importance. These factors, combined with an overall high level of infection in the population, might be responsible for a lower seronegativity rate in our studied male population than in others.

In the current study, seroprevalence of HSV-2 was identical between STD and non-STD clinic attendees. This finding is in contrast to reports from many developed countries, particularly North America and Europe. The differences are most likely attributable to the patient populations from non-STD clinics, i.e., urban municipal hospitals. A considerable proportion of attendees of the pulmonary clinic in Douala have tuberculosis, which is most prevalent among HIV-infected individuals in sub-Saharan Africa. Furthermore, the general hospital involved in this study is open to the public, thus also serving predominantly a population with a low educational and income level. For these reasons, our findings might be specific for the populations in this particular location.

The HSV-2 seroprevalence in Cameroon determined in this study and the reported rates in other countries of tropical Africa 28–32 are higher than the overall seroprevalence in Europe, North America, or other developed countries, even if compared with populations at high risk for HSV-2 infection. 25,33–39 In the United States, however, marked differences in HSV-2 seroprevalence were also found between different racial groups. In the largest, most recent study, seroprevalence rates were 55.1% for black females and 20.2% for white females. 40 For men, rates were 34.7% among blacks and 14.9% among whites. The racial divergences were already present in teenagers and young adults. 27,41,42 In Europe, similar trends have been documented in the United Kingdom. In a study performed with specimens collected in the early 1980s in an antenatal clinic, HSV-2 seroprevalence was highest among black African women (36.5%) but did not exceed 7% among Asian or white women. 43 Concerning the reasons for the racial divergence in HSV-2 seroprevalence, a certain racial predilection for HSV-2 infection cannot be excluded. However, epidemiologic factors of risk populations more likely account for the difference in HSV-2 seroprevalence.

Because most people tend to have sexual contacts within their own ethnic group, the individual's risk of acquiring genital herpes infection is relatively high in populations with high infection rates, even if the number of sexual partners is limited. This aspect would be in agreement with the results of several studies showing that HSV-2 seroprevalence among blacks increases more rapidly with increasing lifetime numbers of sexual partners than among whites. 40,41 In developing countries, additional factors appear to be involved in the widespread dissemination of infection. In a recent study among Kenyan women, it was demonstrated that genital tract shedding of HSV was significantly associated with vitamin A deficiency, thus most probably facilitating sexual transmission of HSV. 31

The high rate of HSV-2 infection in Cameroon poses a significant threat because genital herpes increases the risk of transmitting HIV infection and of becoming infected with HIV. However, HSV-2 serologic testing in Africa provides the options of gathering helpful information about levels of risk for HIV infection, e.g., by identifying highly sexually active populations or gaining insight into sexual behavior at a regional and local level. Moreover, HSV-2 serologic testing may help direct HIV prevention activities and monitor the efficiency of HIV intervention strategies such as education for safer sexual behavior.

In summary, this study provides the first data on the HSV-2 infection rate in three different populations of clinic attendees in Cameroon. Seroprevalence was found to be similar for these three groups and did not differ between males and females. In view of the significant morbidity and mortality caused by HSV-2, there is an urgent need for establishing medical and educational campaigns to reduce HSV-2 infection in order to maintain the sexual and mental health of the population. Because of the accumulating evidence that genital herpes facilitates HIV transmission, these campaigns are of particular importance in Cameroon and other countries in tropical Africa suffering from the devastating consequences of HIV infection.

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