The ethics committees of the Cameroonian Ministry of Health and the Centre Hospitalier Universitaire de Sherbrooke (Sherbrooke, Quebec, Canada) approved the 2009 study and 2010 follow-up specimen testing. The study was conducted in Ebolowa, southern Cameroon (10). Inclusion criteria were age >60 years and consent. Exclusion criteria were dementia or inability to communicate. With cooperation from community leaders, we visited a convenience sample of houses to identify participants. We obtained venous samples from participants and gathered sociodemographic data and information about past intravenous treatment for any disease, past parenteral treatment for infectious diseases, transfusions, scarifications, and circumcision. Vaccine scars were documented.

We performed PARV4 IgG detection on each sample in replicate by indirect ELISA by using baculovirus-expressed viral protein 2 and control antigens (8); arbitrary unit (AU) values were calculated relative to a control sample. Because of a high background reactivity observed for this cohort, we additionally stipulated that for positive samples, the optical density ratio (ODR) of viral protein 2 to control must be >1.2; ODRs below this threshold were considered negative.

Serologic assays for HCV and treponemal antibodies were described in the original study by Pepin et al. (10). We detected antibodies against hepatitis B core antigen (HBcAg) by using AxSYM (Abbott, Montreal, Quebec, Canada) and analyzed data by using Stata 10.0 (StataCorp LP, College Station, TX, USA). Proportions were compared by using either the χ² or Fisher exact test. Variables associated with PARV4 seropositivity in univariate analysis were tested in logistic regression models through nonautomated forward selection, continuing until no other variable reached significance. Each variable was then eliminated to assess its effect by using likelihood ratio tests. We retained in the final model variables that enhanced the fit at the p<0.05 level.

The study comprised 451 persons 60–102 years of age (median 70 years); 56% were HCV seropositive, 74% had antibodies against Treponema (10), and 95% were anti-HBcAg seropositive. Seventy-nine (17.5%) persons carried PARV4 antibodies.

PARV4 antibodies were more prevalent among persons 60–64 years of age than among older persons (Table 1). Prevalence did not vary by sex or by presence of anti-HCV, anti-HBcAg, or treponemal antibodies. The prevalence of anti-PARV4 increased, but not significantly, with exposure to intravenous treatments in general. Receipt of intravenous antimalarial drugs was associated with PARV4 seropositivity, which was also more frequent among persons treated for tuberculosis and among the few women who had received injections of the contraceptive Depo-Provera (Pharmacia & Upjohn Company, New York, NY, USA). PARV4 seropositivity was not associated with treatments delivered by injection against yaws, syphilis, leprosy, or trypanosomiasis (data not shown) or with sexually transmitted infections. PARV4 seropositivity was less common among persons who had a vaccine scar on the left arm.

In multivariate analysis (Table 2), PARV4 seropositivity was associated with younger age, intravenous receipt of antimalarial drugs, and parenteral receipt of antituberculosis treatment (the latter was of borderline significance) and was less common among persons with a left-sided vaccine scar. In that model, Depo-Provera injections were associated with PARV-4 seropositivity among women (adjusted odds ratio 17.27, 95% CI 1.57–189.78; p = 0.02).

To confirm that associations were not biased by assay sensitivity, we conducted a secondary analysis that excluded 81 borderline PARV4-negative persons (AU >0.5 and ODR <1.2) and 35 borderline PARV4-positive persons (AU 0.5–2.0, ODR >1.2) (Table 2). The same factors as in the main analysis were associated with PARV4 seropositivity; receipt of intravenous antimalarial drugs was not significant in the smaller sample.

We retrospectively analyzed samples obtained during a study of elderly Cameroonians from an area where HCV infection was hyperendemic and in which we had collected much information about potential parenteral modes of transmission of blood-borne viruses but less information about other routes (10). Because this was a cross-sectional study, the time sequence of exposure routes and PARV4 infection could not be determined. Thus, our results should be considered exploratory.

The sensitivity, specificity, and ability of our assay to identify seroconversions are comparable to those of PCR-based methods for determining active infections and past exposure (7–9,13). Exclusion of samples showing low antibody levels that might represent nonspecific reactivity had little effect on the analysis of risk factors.

The results provide some evidence for parenteral transmission of PARV4 in the study community. As was HCV infection (10), PARV4 infection was associated with receipt of intravenous antimalarial therapy. This risk factor was found for half of the population we studied, whereas intramuscular Depo-Provera and streptomycin were administered to few patients. In univariate analysis, PARV4 seropositivity was also more common in patients treated with oral antituberculosis drugs. Although the seroprevalence of PARV4 increased with past exposure to intravenous treatments in general, this finding was not statistically significant because antibodies against PARV4 were common among persons who reported no such treatments. This finding, and the lack of association between PARV4 and HCV seropositivity, suggests that other, nonparenteral modes of transmission existed.

PARV4 seropositivity was more common in persons 60–64 years of age than in older persons. This finding has 3 potential explanations. First, exposure to the virus might have fluctuated over time. Second, titers of antibodies against PARV4 might progressively wane, eventually leading to false negative results. Third, PARV4 infection might increase long-term risk for death, although this explanation seems unlikely.

Absence of a vaccine scar on the left arm was associated with PARV4 seropositivity. Historical and epidemiologic data suggest that in Cameroon, the left side was used for smallpox vaccine and the right side for Mycobacterium bovis BCG (14,15). Failure of scar development after smallpox vaccination might reflect immunologic characteristics associated with greater susceptibility to PARV4 infection.

Our findings suggest that some parenteral transmission of PARV4 occurred among elderly Cameroonians, but parenteral transmission might not have been the main route of infection. The association with past tuberculosis, although perhaps coincidental, is intriguing and deserves further study.