The simian origins of HIV-1 and HIV-2 are well delineated [1–3], but the mechanisms that facilitated their simultaneous emergence in different regions of Africa, approximately 50 years ago, are poorly understood. The epidemiology of HIV-2 infection in Guinea-Bissau, its epicentre, remains enigmatic. There is close genetic relationship between HIV-2 and SIVsm, a simian virus infecting sooty mangabeys in the wild, and geographical overlap between their habitat in West Africa and the distribution of HIV-2 . However, HIV-2 is rare in Sierra Leone and Liberia, countries with large sooty mangabey populations, while mangabeys disappeared from Guinea-Bissau [2,3]. Unravelling the factors behind the emergence of HIV-2 might, by analogy, provide insight into the events that led to the HIV-1 pandemic.
It is assumed that HIV-2 jumped species through the manipulation of monkey meat, and later became sexually transmitted [2,3]. Molecular clock analysis suggests that cross-species transmission to humans occurred in Guinea-Bissau around 1940–1945, with exponential growth between 1955 and 1970 . In Bissau and Caio, HIV-2 was much more prevalent in individuals born before 1962 (Table 1) [5–10]. This peculiar age distribution, very distinct from that of HIV-1 elsewhere in Africa, was initially thought to reflect the low pathogenicity of HIV-2, mortality of infected individuals being only two to three times that of seronegatives [11,12]. Subsequent surveys documented a cohort effect, hypothetically attributed to sexual transmission in a generation sexually active during the independence war (1963–1974) [2,6,9]. HIV-2 infection in women was associated with potential proxies for sex work [6,8] (not living with husband, having lived elsewhere in Guinea-Bissau or Senegal, sex with a white man) and high prevalences were found in sex workers .
However, while HIV-1 prevalence in Bissau and Caio increased, HIV-2 prevalence declined [2,9,13,14]. In countries with past colonial ties to Portugal (Angola, Mozambique, Brazil, India), HIV-2 infections were reported 20 years ago but then disappeared [2,13]. In Senegal, heterosexual spread of HIV-2 among sex workers was notably slower than for HIV-1 . These observations suggest that its basic case reproductive number is < 1 when HIV-2 is transmitted sexually, in line with its modest genital shedding [2,16,17]. But then, how did prevalence reach such high levels in the first place? Transmission of hepatitis C virus during campaigns for schistosomiasis control in Egypt demonstrated that iatrogenic epidemics can reach a massive scale . Others have hypothesized that parenteral transmission of HIV-2 (through injections, vaccinations, transfusions and traditional practices) must have been the predominant route in Guinea-Bissau , and that campaigns for sleeping sickness (trypanosomiasis) control in Central and West Africa contributed to the emergence of HIV-1 . To investigate these hypotheses, we conducted a study among the generation of Bissau-Guineans belonging to the high HIV-2 prevalence cohorts.
The study was ethically approved by the Guinea-Bissau Ministry of Health and the institutional review board of the Centre Hospitalier Universitaire de Sherbrooke, Canada. Inclusion criteria were age ≥ 50 years and willingness to consent. Exclusion criteria were dementia, inability to communicate or understand one of the languages spoken by the interviewers. Participants were recruited, in January–March 2005, from the following areas of Bissau: Pluba, Cupelom, Luanda, Santa Luzia, Penha, Pefine, Calequir, Bairro Militar, Amedalai, Rossio, Antula and Reno. Compounds were visited to identify individuals aged ≥ 50 years. If consent was given, a questionnaire was administered in a private setting, gathering data on sociodemographic characteristics, past medical histories, exposure to monkey meat, and a limited number of questions on sexual behaviour. For participants unable to provide their exact age, an estimate was made based on historical events widely known locally. A finger prick was performed and capillary blood deposited on a filter paper. Questionnaires and filter papers were identified only by a study number. As a compensation for participation, interviewees were given antihelminthic drugs and antimalarials, to be used when needed. Most visits were done during daytime, or early in the evening in an attempt to recruit male participants. As our aim was to measure associations between HIV-2 and specific events rather than its exact prevalence, this was a convenience sample. We did not formally measure it, but refusal to participate was very uncommon especially among women.
Blood specimens were blotted on a Whatman 3 filter paper, air dried at ambient temperature and stored in plastic zip-closure bags at 4°C for 1–2 months before testing. A 6-mm dried blood spot (DBS) was punched and eluted in 250 μl of sample diluent (Detect-HIV: Adaltis, Montreal, Quebec, Canada) for 16 hours at 4°C. HIV serology was performed using Detect-HIV with 100 μl of eluted sample. Non-reactive samples were considered HIV-seronegative whereas reactive samples were tested with Genie II HIV-1/HIV-2 (Bio-Rad Marnes la Coquette, France). Genie II dually reactive samples (to HIV-1 and HIV-2) and discordant samples (Detect-HIV reactive/Genie II non-reactive) were further tested by INNO-LIA HIV I/II Score (Innogenetics, Gent, Belgium) using 1 ml of eluted sample (obtained from two DBS in 1.2 ml of INNO-LIA sample diluent).
Data were analysed with Stata 8.0 (College Station, Texas, USA). Proportions were compared with the chi-squared test, or Fisher's test when appropriate. Logistic regression was used to calculate adjusted odds ratios (AOR) and their 95% confidence intervals (CI). Models were built up sequentially, starting with the variable most strongly associated with the outcome and continuing until no other variable reached significance. When the final model was reached, each variable was dropped in turn to assess its effect. Different models were compared using the likelihood ratio test, finally keeping variables significant at the P = 0.05 level.
Out of 1608 participants, 184 (11.4%) were infected with HIV-2, 48 (3.0%) with HIV-1, 21 (1.3%) had dual HIV-1/HIV-2 infections and two had an indeterminate serology. Prevalence of HIV-2 was higher in women (143/1063; 13.5%) than men (41/545; 7.5%) (P < 0.001). No such differences were seen for dual HIV-1/HIV-2 infections [17 (1.6%) women; four (0.7%) men, P = 0.22] and for HIV-1 [35 (3.3%) women, 13 (2.4%) men, P = 0.39]. Subsequent analyses considered those participants infected with HIV-2 only as well as those whose serology indicated a dual HIV-2/HIV-1 infection, and compared them to the seronegatives. As there were sex-specific risk factors (excision, circumcision), results were initially stratified by sex.
HIV-2 prevalence was higher in women aged 50–59 years, and in those widowed (Table 2). Prevalence tended to vary between ethnic groups, and was highest amongst the Fulas. Other factors significantly associated with HIV-2 in univariate analyses included having received injections for the treatment of tuberculosis and having undergone ritual excision. HIV-2 was not associated with having received injections of pentamidine for the prevention of trypanosomiasis, nor with the number of years spent in Portuguese-controlled territories during these pentamidine campaigns, or with having fought in the guerrilla movement. Very few women admitted having sold sexual services, but there was no association between HIV-2 and variables previously considered proxies for sex work (travel outside the country, sex with a white man). In multivariate analysis, the independent risk factors for HIV-2 infection were age, marital status, having been excised and having received injections for the treatment of tuberculosis. No interaction was found. In a sub-analysis that excluded the 17 women with dual HIV-1/HIV-2 infections, the same independent risk factors were identified (data not shown).
Thirty-five women were infected with HIV-1 only. HIV-1 was more common among women aged 50–59 years (27/495; 5.5%) than in those ≥ 60 years old (8/501; 1.4%) (P < 0.001). HIV-1 was not associated with excision, injections for the treatment of tuberculosis or trypanosomiasis, other parenteral exposures, marital status nor ethnic group (data not shown).
The distribution of HIV-2 was remarkably homogeneous (Table 3). Most notably, it did not vary whether men reported a past history of urethral discharge, genital ulcer, or contacts with sex workers, nor whether they fought for the guerrilla movement or the Portuguese army during the independence war. Thirteen men were infected with HIV-1 only. Prevalence of HIV-1 infection was 3.0% (7/233) in men aged 50–59 years and 1.9% (6/311) in those ≥ 60 years old.
Women and men combined
In an analysis regrouping men and women (and which, for that reason, did not consider excision nor male circumcision), HIV-2 was significantly associated with having received injections for the treatment of tuberculosis (AOR, 2.12; 95% CI, 1.11–4.05; P = 0.02) or for the treatment of trypanosomiasis (AOR, 1.75; 95% CI, 1.03–2.97; P = 0.04), with being aged 50–59 years, female sex, being widowed and being a Fula (Table 4). HIV-2 was not associated with immunization scars, traditional scarifications or blood transfusions (data not shown). No interaction was found. After excluding Fulas or Mandingas (who do not manipulate or eat monkeys, on religious grounds), no association was seen between HIV-2 and having hunted, cut or eaten monkey meat (data not shown).
Our results suggest that parenteral transmission through at least three mechanisms played a role in the dynamics of HIV-2 in Guinea-Bissau decades ago. First, HIV-2 infection was associated with having received injections of streptomycin for the treatment of tuberculosis. In Guinea-Bissau, HIV-infected patients with tuberculosis have been treated with a regimen comprising only oral drugs since 1992 . The 61 individuals who received injections for the treatment of tuberculosis were thus treated more than a decade ago. In Guinea-Bissau and The Gambia, mortality after diagnosis of tuberculosis in HIV-2-infected patients was 45–53 per 100 person-years (median CD4 cell count at diagnosis: 104–225/μl) [21,22]. Thus, it appears unlikely that the association between injections of streptomycin and HIV-2 infection merely reflected HIV-2-induced immunosuppression and the attending risk of developing tuberculosis. Indeed, the use of streptomycin in the treatment of tuberculosis offered a unique opportunity for the parenteral transmission of HIV-2, as these patients received at least 60 intramuscular injections, usually on consecutive days.
Second, during the colonial era Guinea-Bissau experienced a high incidence of trypanosomiasis, peaking at 2169 cases in 1952; relapses were common so that, in 1953, 3229 patients (total population of the country: approximately 500 000) received 37 509 injections of trypanocidal drugs . Incidence progressively decreased and the disease ultimately disappeared, following a mass campaign of chemoprophylaxis with intramuscular pentamidine given half-yearly to two-thirds of the population of Portuguese-controlled areas, from 1965 until independence [23–25]. We found no evidence that prophylactic injections of pentamidine contributed to HIV-2 transmission. However, the 104 men and women who developed trypanosomiasis and received trypanocidal drugs were more likely to be HIV-2-infected, presumably reflecting parenteral transmission during the administration of intravenous arsenical drugs (up to 15 injections, later repeated if a relapse was documented) or, for those with early-stage trypanosomiasis, intravenous suramin (approximately five injections) or intramuscular pentamidine (approximately 10 injections).
Third, there was an association between excision and HIV-2, explaining the higher prevalences among Fula and Mandinga women (97% of whom were excised). Partial or complete clitoridectomy might facilitate the sexual transmission of HIV-2 during somewhat traumatic intercourses but, to our knowledge, this co-factor effect has never been demonstrated for HIV-1 [26,27]. In sex workers in The Gambia and Senegal, high HIV-2 prevalence (10–38%) groups among whom most of the transmission must have been heterosexual, excision was not associated with HIV-2 [28,29]. A more likely scenario is that HIV-2 transmission occurred during the fanado, an initiation ritual through which dozens of girls (generally aged 8–12 years) [30–32] were excised on the same day by ngamanos, elderly women using the same ceremonial knife on a highly vascular organ. HIV-2 might have been introduced into these cohorts by girls infected occupationally, parenterally or vertically . A prior study in Bissau reporting no association between HIV-2 and ‘female circumcision’ had little power since only 7% of women were not excised, whereas in Caio (where many had been sex workers) only 1% of women were excised [10,34].
HIV-2 was not associated with traditional scarifications, BCG or smallpox immunization scars, nor with transfusions. Previous reports about transfusions have been contradictory [6,8,10]. In Bissau, donated blood has been screened for HIV since the late 1980s; over time, the proportion of past transfusions that had not been unscreened has decreased. Individuals with transfusion-associated HIV-2, for whom the infectious dose was massive, might also experience a higher mortality .
We found no association between manipulation of monkeys (their hunting, cutting or eating) and HIV-2. Given that sooty mangabeys disappeared from Guinea-Bissau , such activities presumably led to cases of occupational HIV-2 infection in the distant past, potentially initiating chains of transmission, but cannot explain an epidemic of the magnitude seen in Guinea-Bissau, which had to be amplified by other routes. The high HIV-2 prevalence among Fula and Mandinga women, Muslims who refuse to manipulate or eat monkeys, also argues against this mode of transmission having been substantial.
Among men, HIV-2, as in most previous studies [6–8,10,34], was not more common in those with a past history of urethritis or genital ulcer, nor among men who used the services of sex workers, something highly unusual for a sexually transmitted infection. Some men must have been infected parenterally: power was limited, but odds ratios for associations between HIV-2 and treatment of tuberculosis or trypanosomiasis were similar to those seen in women. Furthermore, as 97% of men were circumcised, we could not verify whether this procedure was associated with HIV-2, but the above-mentioned infection control issues are also relevant for ritual male circumcision . HIV-2 prevalence did not vary according to involvement in the armed conflict preceding independence. The hypothesis that conflict-related changes in sexual behaviours led to the peculiar age distribution of HIV-2 in Guinea-Bissau  seems implausible for several reasons: it would have required sexual behaviours to change dramatically immediately after the war, few Portuguese soldiers stationed in the cities acquired HIV-2 , and potential contacts with sex workers must have been limited for men in the liberation movement who fought a guerrilla war.
In the analysis that combined men and women, younger age (50–59 years) was associated with HIV-2 infection. If parenteral transmission occurred during some period of time and then stopped or was reduced drastically, the cumulative HIV-2-associated mortality will eventually result in a lower prevalence in older individuals. Female sex was also associated with HIV-2, a finding which was not completely explained by transmission during excision, as the higher prevalence in women in comparison with men persisted after excluding women who had been excised, or after excluding Fulas and Mandingas. A higher susceptibility of older women to heterosexual transmission of HIV-2 has been suggested, but was based on few outcomes [9,35]. A longer survival among HIV-2-infected women in comparison with men has been observed in a large clinic-based cohort in The Gambia , and would also explain the higher prevalence among female widows.
Study limitations include its cross-sectional nature (time sequence of events is uncertain), the lower recruitment of males, and possible recall biases when participants were asked to report events that might have occurred decades earlier. Even if less lethal than HIV-1, HIV-2-infected individuals have a mortality that is two to three times higher than seronegatives. Over a very long period of time, this will bias towards the null value the strength of the associations between HIV-2 and exposures and underestimate the prevalence of some exposures. For these reasons, we did not calculate population attributable-risk fractions. The use of the INNO-LIA assay might have led to an overestimation of the number of dual HIV-1/HIV-2 infections . However, the findings were identical if analysis was restricted to those with HIV-2 infection only.
The following scenario seems plausible: for a long time, HIV-2 infected some individuals who hunted or prepared sooty mangabey meat, with little subsequent transmission. Then, the virus spread parenterally starting in the late 1940s, through needles, syringes, and instruments used for excision. The high incidence of trypanosomiasis and the availability of streptomycin for tuberculosis patients facilitated this phase, which might have led to adaptive mutations of the virus through serial passages . We documented three routes for parenteral transmission, but it is possible that others existed. Sexual transmission then occurred but rather ineffectively, except between spouses having repeated intercourses over many years and among sex workers with thousands of clients. The few cases in countries with a colonial link to Portugal might have resulted from the sexual or parenteral transmission to Portuguese nationals, or perhaps the distribution of contaminated blood products out of Portugal. HIV-2 is slowly disappearing because the opportunities for parenteral transmission have been reduced: trypanosomiasis was eliminated, streptomycin replaced by oral drugs, injection practices are safer, donated blood is screened, and excision is practiced differently, at least in urban areas where the cutting (fanadozinho, little initiation), now divorced from the wider rituals (fanado garandi, big initiation), is done in the first 5 years of life, often individually and less publicly than before .
It remains unclear why there has been no HIV-2 epidemic in Sierra Leone and Liberia, natural habitats of the sooty mangabey, where excision was common and streptomycin introduced simultaneously as in Guinea-Bissau. There was little trypanosomiasis in the former countries; perhaps large-scale use of intravenous trypanocidal drugs was necessary to jumpstart the epidemic. Alternatively, HIV-2 subtype A, the only one in Guinea-Bissau, might be intrinsically more prone to parenteral transmission than the more diversified subtypes found in Sierra Leone and Liberia [2,39,40].
In recent years, some researchers have argued that parenteral transmission is the main mode of transmission of HIV-1 in Africa nowadays . We do not share this view at all: there is overwhelming evidence, summarized elsewhere , that currently the main route of HIV-1 transmission is sexual intercourse, on which prevention programs must focus. Our study attempted to delineate the events that led to an epidemic of HIV-2 in Guinea-Bissau in a generation presumably infected 20–50 years ago. It was made possible by the prolonged survival of some HIV-2 infected individuals, and was driven by the belief that understanding the past can provide wisdom useful to future generations. Although the same events could certainly have initiated chains of HIV-1 transmission in Central Africa [20,43], as supported by mathematical modelling , the most unfortunate difference between the two human-infecting retroviruses is that the basic reproductive number (R0) of HIV-1, when transmitted sexually, is greater than one.
We are indebted to Dr Inacio Alvarenga (World Health Organization, Bissau) and Dr Francisco Dias (Ministério da Saude Publica, Guiné-Bissau) for their assistance; to Dr Michelle C. Johnson (Bucknell University) for generously sharing information on excision practices in Guinea-Bissau; to Dr Shabbar Jaffar (London School of Hygiene and Tropical Medicine) for critical review of the manuscript; to our field workers Arlinda Alvarenga, Mari Turé, Isis Ferreira, Inàcio L. Vieira, Cidia Camarà, Maria Domingos; and to all study participants.
Conflict of interest statement: The authors have no conflict of interest to declare.
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