Since 1986, when the first case of acquired immunodeficiency syndrome was identified in Lesotho, a small country in Southern Africa, the number of HIV infections has risen exponentially. Today, the number of people living with HIV in Lesotho is estimated at 270,000 of a total population of around 2 million people.1 It has been hypothesized that the Lesotho Highlands Water Project resulted in the influx of a migrant workforce composed of predominantly single males into a relatively isolated, mountainous area where HIV was previously unknown.2 Currently, the HIV seroprevalence in adults aged 15 to 49 is estimated to be 23.2%. However, access to antiretroviral therapy (ART) has improved substantially over the past years and ART coverage is now estimated at 26%.1
HIV-infected people are often coinfected with other, often “opportunistic” viruses, especially if living under conditions of poverty, crowding, and poor hygiene. Such coinfections may influence the outcome of ART. Little is known so far on the prevalences of these coinfections in Lesotho.
Herpes simplex virus Type 2 (HSV-2)-induced genital lesions are regarded as an important cofactor for HIV transmission.3 Just as HIV, HSV-2 is predominantly sexually transmitted. Its prevalence is, in general, higher in Africa when compared to other continents,4 and HIV-seropositive cohorts tend to show higher anti-HSV-2 prevalences (>70%) than HIV-negative ones (50%).4
Cytomegalovirus (CMV) is an ubiquitous herpesvirus, transmitted in utero, through breastfeeding and horizontally through close contact and sexually throughout life. CMV seroprevalence rates in the adult population worldwide are between 60% and 100%5–7 and may be >93% among healthy blood donors in African countries.8
With the decreasing mortality from acquired immunodeficiency syndrome-related opportunistic infections after the introduction of ART, liver disease related to hepatitis B virus (HBV) or hepatitis C virus (HCV) infection has emerged as an important cause of morbidity and mortality in the coinfected population.9 Although HBV prevalence varies widely across the African continent, 70% to 95% of the population have been exposed to HBV infection, of whom 8% to 20% are estimated to be HBV carriers.10–12 Of the 400 million chronic HBV carriers worldwide, 50 million are estimated to reside in sub-Saharan Africa.9 More than 170 million people (around 3% of the world's population) are chronically infected with the mostly blood-borne HCV.9 Available data indicate a low prevalence of HCV infection and also of HCV/HIV coinfections in most parts of sub-Saharan Africa.13
In contrast, the seroprevalence of the water- and food-borne hepatitis A virus (HAV) is very high (>99%) in most sub-Saharan African populations.14
In different studies from southern Africa, pregnant women had reactive syphilis serology in 9.2% in Zimbabwe in 1998 and 12% in rural South Africa in 2000.15,16 Among young adults in a rural Gambian community, 10% of women and 2% of men were seropositive.17 This gender-specific difference was also described in other studies, for example, in South African gold mining community (18.7% in females and 8.1% in males) in 2000.18 In contrast to the aforementioned viral infections, this sexually transmitted spirochete is well treatable.
No seroepidemiological data are hitherto available on the prevalence of viral coinfections in HIV infected individuals in Lesotho. We, therefore, investigated the seroprevalence of the mainly faeco-enterally transmitted HAV, the sexually transmitted HSV-2 and Treponema pallidum (T. pallidum), the vertically and horizontally transmitted HBV and CMV, and the blood-transmitted HCV in a cohort of antiretrovirally treated HIV-infected individuals in Mafeteng, Lesotho, who attended the ART treatment program at a District Hospital in the Lowlands of Lesotho between January and March 2007. In addition, we investigated the serological response to HSV-2 and to T. pallidum semiquantitatively in correlation with the patients' immunologic status as assessed by CD4 cell count.
MATERIALS AND METHODS
Patients and Specimens
HIV-infected outpatients who had received ART for at least 1 year at the Karabong Clinic of Mafeteng Government Hospital, Lesotho, were enrolled in this study. Patients were approached for study participation on a first interview during their routine visit to the clinic. Potential and voluntary study participants received detailed information in Sesotho and English in respect to the content of the study and had at least 24 hours to decide on study participation. In the case of patients being illiterate, the informed consent form was read loudly in presence of 2 witnesses. Questions to test comprehensions were then posed, and the patient and the witnesses signed the informed consent.
The patients were considered eligible for study participation if they had signed the informed consent, were 15 years of age or older, registered for ART, and initiated ART at Karabong Clinic before March 5, 2006. The patients meeting the following exclusion criteria were not considered: unable or unwilling to give informed consent, less than 15 years of age, any previous ART taken before initiation of ART at Karabong Clinic with the exception of Nevirapine as intrapartum transmission prophylaxis, or transferred in from other centers providing ART. The period of recruitment was more than 4 months.
In total, 205 subjects were enrolled between January and March 2007 and had samples collected for the study. These included 53 men and 152 women; median age was 42 (range, 28–71) years for males and 37 (range, 19–64) years for females. For further analysis, patients were grouped into age groups [19–29 (n = 18), 30–39 (n = 71), 40–49 (n = 67), 50–58 (n = 34), 61–71 (n = 6) years]. The patients participating in “patients in retroviral therapy” (PIMA) started HIV therapy with a severe immune defect represented by the World Health Organization (WHO) classification at treatment initiation: 82% of women (124/151) and 90% of men (47/52) were at WHO stage III or IV (3 female patient data missing), indicating a clinically relevant immunodeficiency as an indicator for treatment initiation. Of those patients where CD4 count before treatment initiation was available (n = 196), 92% of men and 89% of women had a CD4 count less than 200 cells/mm3. Treatment success and at least partial immune reconstitution was visible at the clinical study visit taking place after 1 year of treatment initiation (range, 358–867 days), where all patients presented with a Karnowsky Index of 95.5% (male, 95.3% [range, 70%–100%]; female, 95.5% [range 70%–100%]). All patients received dual-class ART with 2 NRTI, and 1 NNRTI, of which most (97%; n = 199) received Stavudine, Lamivudine, and Nevirapine as first-line regimen.
The PIMA study population was taken from a total of 2309 patients who had been enrolled into the national antiretroviral program at Karabong clinic up to March 2007. Of those, 744 were men (32%), and 1555 women (67%), in 10 patients, the gender could not be identified from chart review. Of them, 893 were on therapy, 311 men and 575 women.
Ethics clearance to undertake this study had been obtained from the Ethics Committee of the Ministry of Health of the Kingdom of Lesotho before study initiation. The protocol and all respective study documents were also approved by the Ethics Committee of the University Hospital of the Goethe University Frankfurt am Main, Germany (reference number: 43/07). Data were generated in line with the principles of Good Clinical Practice using an anonymized case report form.
CD4 cell counts were performed at the Mafeteng Hospital laboratory. Thereafter, the remaining EDTA plasma was collected, frozen on dry ice, and transported to the Institute for Medical Virology, Frankfurt University Hospital, for further testing.
CD4 cell counts were determined by FACS analysis BD FACS Count (Becton, Dickinson and Company) at the central laboratory of Mafeteng Government Hospital.
Commercially available enzyme-linked immunosorbent assays were used: Anti-HAV and anti-HBc were analysed by AxSYM MEIA, anti-HCV, hepatitis B surface antigen (HBsAg), anti-CMV, and T. pallidum antibodies by Architect CMIA (both Abbott, Wiesbaden, Germany). The T. pallidum test indicates active as well as past infection.
HSV-2-specific antibodies were analyzed by the Liaison HSV-2 assay (DiaSorin, Dietzenbach, Germany). This assay is based on recombinant gG2 antigen and allows type-specific detection of IgG antibodies against HSV-2. All tests were performed according to the manufacturers' specifications.
Values for HSV-2 antibodies were measured as AU/mL (arbitrary units/mL), values for T. pallidum antibodies as sample/cutoff (s/co) ratio. Since all values for HAV and CMV antibodies yielded overflow optical density values and no dilutions were tested, no quantification was made. HBsAg was determined as IU/mL.
HCV RNA was measured quantitatively by using the COBAS AmpliPrep/COBAS TaqMan System (Roche Molecular Diagnostics; Mannheim, Germany).
Statistical analyses were performed with the Statemat program (GraphPad, San Diego, CA) using the Wilcoxon signed rank test. Significance was assumed if P < 0.05; 95%confidence intervals (CI) for proportions were calculated using the program BiAS for Windows 8.3 (Epsilon Press, Hochheim, Darmstadt, Germany 2007).
Patients of our cohort had started ART at least 1 year before the study visit and generally received dual class triple drug regimen according to the national treatment guidelines of Lesotho, which is aligned with the WHO public health approach 2003 revision. Consequently, most of the patients were on Lamivudine plus Stavudine combined with either Efavirenz or Nevirapine. As the national ART program in Lesotho was only started in 2004, these patients represent the first recipients of ART through the national program.
The patients showed an average CD4 cell count/μL of 390 (range, 32–1108) in women and 308 (range, 83–991) in men. There was a nonsignificant trend of a decrease in CD4 cell count with increasing age. In the male age group of 30 to 39 years, the mean cell count was 312 CD4 cells/μL; in the 40 to 49 group, 313; and in the group of 50 to 59 years, 302; in the same female age groups it was 411, 372, 319 cells/μL, respectively.
For HAV and CMV very high seroprevalences of 98.5% and 100%, respectively, were found. In contrast, only 1 of the 205 subjects (0.5%) had HCV antibodies. This 32-year-old male patient had a HCV viral load of 2.2 million IU/mL. No information was available on the history of his HCV acquisition.
Although HBV exposure (anti-HBc positive) had occurred in 35.5% of the patients, only 5.4% of the exposed individuals were positive for HBsAg indicating active infection (Table. 1). Anti-HBc positivity showed no significant differences between age groups or between females and males.
For the sexually transmitted agents, an overall prevalence of 78.5% was found for HSV-2 compared to 29.0% for syphilis. Analysis of the age groups revealed no significant prevalence differences between genders for either.
To compare the immunologic responses to HSV-2 and T. pallidum, the specific antibody reactivities were analyzed quantitatively. When females and males were compared within the age group 30 to 39 years, the median anti-HSV-2 titer (in AU/mL) was 430 in females and 324 in males (P = 0.065); in the age group 40 to 49, 480 in females and 366 in males (P = 0.066); and in the age group 50 to 58, 439 in females and 390 in males (P = 0.48, not significant), respectively (Fig. 1). Because in the last age group, 61 to 71 years, only 4 females and 2 males were included, data were not analyzed for significance. The differences in anti-HSV-2 titers in the age groups 30 to 39 and 40 to 49 are close to being significant and may be indicative of a stronger immune response in females.
In contrast to HSV-2 titers, the antibody levels against T. pallidum did not differ significantly between males and females (Fig. 2) and between age groups.
When anti-HSV-2 titers were analyzed in patients grouped according to CD4 cell count, amongst those with >500 CD4 cells/μL females showed a slightly higher anti-HSV-2 titer than males with 447 to 398 AU/mL, respectively (P = 0.87). In those between 250 to 500 CD4 cells/μL, median anti-HSV-2 values were 467 AU/mL for females and 302 AU/mL for males, showing a significant difference (P = 0.001). In T-cell depleted patients with <250 CD4 cells/μL at study visit, no gender-specific difference could be detected (490 AE/mL in females and 425 AE/mL in males; P = 0.36) (Fig. 3). Thus there was a better lasting immune response against HSV-2 in females despite the decreased CD4 cell number of 250 to 499/μL and thus a marked gender difference. When the groups were divided in CD4 cell count/μL as <200, 201 to 349, and 350 to 499 between females and males in the <200 group there was no significant difference (P = 0.64), whereas in both other groups there was a significant difference of P = 0.035 for 201 to 349, and of P = 0.002 for 350 to 499 calculated for the median. Similar results were obtained calculating data using the median—P values were P = 0.58; P = 0.02, and P = 0.002 for the 3 groups, respectively. Thus in our female study population there seems to be a higher stimulus for HSV-2 antibody production.
This study was designed to determine the seroprevalence of viral coinfections in ART-treated HIV-infected individuals in a rural area of Lesotho.
Our data show that the seroprevalences of HAV, HBV, and CMV are comparable to those previously reported from other Southern African countries.19–21 Children in sub-Saharan Africa show an anti-HAV prevalence of approximately 97% with the age of 14 years22 and nearly 100% with 20 years.14,23 The high anti-HAV prevalence found in our study (98.5%) is in accordance with available epidemiologic data for Southern Africa, and since this infection is mostly acquired during early childhood with continuous subsequent re-exposure, it is plausible that seroprevalence is not influenced by HIV infection.
Different African studies demonstrate that HBV prevalences varies widely, with positivity rates of 8% to 20% for HBsAg and 70% to 95% for anti-HBc.10–12,14,23 HIV/HBV coinfection rates have been studied in Africa with conflicting results, with both higher and lower rates of HBV in HIV-positive patients.9 For example in a cohort of HIV-infected Nigerians the HBsAg seroprevalence was 11.9%.24 Another study from Nigeria showed an increased prevalence of HBsAg in HIV-infected patients (25.9% in HIV-positive patients vs. 14.9% in HIV-negatives).25,26 Results from a cohort of HIV infected individuals in South Africa who had received at least 27 weeks of ART showed a HBsAg prevalence of 19.8%.27 Nevertheless, studies describe “occult” HBV infection in up to 23% of HBsAg-negative, anti-HBc-positive HIV-infected patients who have detectable blood HBV-DNA in South Africa.28 The general exposure in adults to HBV, determined by anti-HBc, was similar for HIV-positive and negative patients in South Africa, with 82% and 85%, respectively.29 A statistically significant difference in the prevalence of HBV-DNA in the HBsAg-negatives has been described between the 2 groups: 33.3% (5/15) of sera with the serological pattern “anti-HBc alone” were HBV DNA-positive HIV-positive group.29
The prevalence markers for HBV in South Africa29 are higher than those reported in our study (35.5% anti-HBc positive, 5.5% HBsAg positive). The reason for this difference might be the difference between an urban and a rural population. As no HIV-negative control group was selected for Lesotho, it is difficult to argue whether the low HBV detection rate can be attributed to a different prevalence in rural Lesotho compared to urban South Africa.
Nevertheless, comparing the relation of active HBV carriers in the cohort of anti-HBc positives, which is between 10% to 20% in the aforementioned studies,10–12,14,23 the data of our study are in good agreement (5.5% HBsAg carriers and 35.5% anti-HBc positives), resulting in a relation of 1:6.5 (or 15.4%).
The anti-CMV prevalence in our cohort was 100%, a result that closely matches with 93.2% among healthy Ghanaian blood donors and 98% in young adults in Cameroon.8,30 In another study from Ghana, there was no statistically significant difference in seroprevalence of CMV between HIV-positive patients and HIV-seronegative healthy blood donors.31 Our data support the observation that there seems to be no relevant difference in the high CMV seroprevalence in HIV-positive or HIV-negative sub-Saharan people.
In contrast to the high seroprevalence rates of HAV, HBV, and CMV, the prevalence of HCV infection across sub-Saharan Africa is much lower and estimated at 5.3%, with a wide variation across the continent (e.g., 7.4%–18% in Egypt, 6% in central sub-Saharan Africa, and 1.6% in Southern and Eastern Africa). Data on HCV/HIV coinfection are very scarce; nevertheless it appears that the prevalence of HIV/HCV coinfection is low in most parts of Africa,13 which was also a result of our study (0.5%; 95% CI: 0.0%–2.8%). This is a remarkable difference to the European and American situation. Although in a cohort of HIV-infected Nigerians anti-HCV was present in 4.8%,24 in a study from South Africa from the KwaZulu-Natal province, which is close to Lesotho, there was a significantly higher prevalence of HCV among HIV infected patients as compared to HIV negatives (13.4% vs. 1.7% respectively; P < 0.001).32 Because the main primary modes of transmission of HCV and HIV infections are different (blood-borne vs. sexual), the very low anti-HCV prevalence rate in our study population might be an indicator that HIV transmission by blood transfusion and intravenous drug use is uncommon in Lesotho and that sexual transmission of HIV is predominant.
The seroprevalence for T. pallidum-specific antibodies in our study was 29.0% (95% CI: 22.8%–35.8%). These data are much higher than those presented in a cross-sectional study among HIV-infected (7.3%) and -uninfected (2.6%) antenatal attendees in an African multisite clinical trial in Malawi, Tanzania, and Zambia in 2006.33 No data are available on whether our patients had been treated for syphilis. A high prevalence of syphilitic lesions facilitates transmission of HIV through mucosal ulcerations.
A higher prevalence of HSV-2 in women compared to men has been reported in different studies from around the world.34–38 Most of the studies also confirm an age-dependent increase. HSV-2 prevalence is, in general, very high in Africa when compared with other continents.4,38 Prevalence varies between 80% in women from Northwestern Tanzania in 2007,39 69.3% in South African blood donors in Durban in 2008,40 and 53% in Zimbabwe in HIV-negative women in 2007.41 Thus the general prevalence of 78.5% (Table 1) found in our study is still in the expected range of the increase of exposure to HSV-2 with increasing age.42 Such high rates are found only in high-risk groups in Europe.34,37,38,43
A new result of this study is the finding of higher anti-HSV-2 titers in still immunocompetent females, based on CD4 cell count. In this context it has to be taken into account that CD4 counts can vary widely (e.g., 265–1932 cells/μL in HIV negatives in Ghana44) and that CD4 baseline may change from given health, gender, and economically related disparities and from region to region.44 Although the WHO reports that normal CD4 counts range from 500 to 1500 cells/μL, a lower CD4 cell count must not be associated with immunodeficiency. In our study only when the CD4 cell count was between 250 and 500 CD4 cells/μL the difference was significant (P = 0.001) between both genders. In the patient group with CD4 cell count >500 CD4 cells/μL the number of females and males were 49 and 4, respectively, suggesting that both groups are hardly comparable. When total CD4 cells were analyzed between females and males values were again significantly higher in females (390 in females and 309 CD4 cells/μL in males; P = 0.0001). A higher antibody response in women against an experimental HSV-2 vaccine has been observed previously.45,46 It has also been reported from mouse experiments that progesterone increases susceptibility to HSV and decreases the immune reaction.47,48 Thus one might argue that after the progesterone cycle in women under estrogen influence the immune response to HSV-2 is enhanced and thus results over time in a higher or rebound antibody response, as found in this study. Apart from the immune status, females in the age groups 30 to 39 and 40 to 49 years showed an essentially higher median anti-HSV-2 titer than males in our study (Fig. 1), 430 versus 324 and 480 versus 366 AU/mL, respectively, despite of just not being significant with P values of 0.065 and 0.066.
In this cohort of HIV-infected patients in Mafeteng in rural Lesotho, seroprevalences of HAV and CMV were found to be very high and equal in both genders. HCV infection was found in only 1 case. HBV and T. pallidum exposure were seen in around one-third of the patients. HSV-2 prevalence was high and showed gender-specific—females higher than males—CD4 cell count- and age-dependent differences in the titer of HSV-2 antibodies, which were not observed for syphilis or anti-HBc. Further studies are needed to analyze if there is a clinical relevance in quantitation of HSV-2 antibodies and whether this observation will be found in other cohorts. Our evidence of a high prevalence of STDs such as syphilis, HBV, and HSV 2—and perhaps further undiagnosed STDs—may contribute to explain the rapid increase in HIV prevalence in Lesotho since 1986.
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