The studies we describe were performed as an adjunct to the investigation of an outbreak of MHF in May 1999. The epicenter of the outbreak was the village of Durba in the Haut-Uélé District, Oriental Province, in northeastern DRC, an isolated region approximately 200 km from the borders of Uganda and Sudan (Figure). Although no official population count for Durba is available, unofficial estimates are approximately 25,000. Watsa, a larger town of approximately 60,000 and the administrative seat of the zone, lies 14 km away. Although the Yogo ethnic group predominates, the population of Durba/Watsa is quite heterogeneous, as many people have migrated to the area to work in the local gold mines. Most are Catholic. The area has had intermittent armed conflict since the beginning of the Congolese civil war in 1996, a situation that has severely limited travel and economic growth.

The livelihood of most of the population in the Durba/Watsa area is associated with gold mining, conducted almost exclusively by young men and most often without professional training or equipment. Some older men, women, and children are involved in the extraction of gold from ore and its sale. Subsistence farming and hunting are also common. Although various mines exist in the area, most mining appears to take place in the Goroumbwa mine a few kilometers from the village of Durba. In addition, some miners dive in local rivers in search of gold. The existence of a hemorrhagic illness in the region appeared to be common knowledge and was labeled “Durba hemorrhagic syndrome” or “Durba syndrome” by the villagers, who often associated it with working in the mines.

Because of the remoteness of the region and the war, supplies are severely limited in all the health facilities in the region. The major facility in Durba is a small rudimentary government health center staffed by a few nurses. In Watsa, there is a larger government hospital, a hospital affiliated with the mining company, and two government health centers. In addition, at least 14 small private health centers operate collectively in Durba and Watsa.

Two surveys were undertaken. The first was a cross-sectional survey on a convenience sample of the general population of Durba township. It was performed by establishing a post in the center of the village. With the aid of local HCWs and a village loud-speaker system, residents of sequential “quartiers” of the village were requested to come for evaluation over a 3-day period. Persons <15 years of age were excluded. The second survey focused on HCWs at all health centers in Durba and Watsa. All HCWs were surveyed at their place of employment.

The rationale for conducting the study was explained to all participants, and verbal consent was obtained. Questionnaires were pretested on local villagers not included in the final study. For the general population, a 2-page questionnaire was administered. Supervisors at the local mining company were consulted about appropriate questions regarding exposures in the mines. Persons were first evaluated by local village HCWs, who determined their ability to speak and understand French. If deemed able to do so, the person was then interviewed by either a local HCW or a French-speaking member of the international investigative team. For those persons who did not understand French, the questionnaire was administered by a local HCW in the appropriate local language. Participants were asked about their entry into and activities within the mines, exposures to persons presumably sick with Durba syndrome (defined as a severe illness with high fever and bleeding from the nose, mouth, and/or anus) in the hospital and at home, and exposures to various animals thought to possibly transmit Marburg virus (rodents, bats, and monkeys). Participants were given a small bag of peanuts as a token of appreciation for their cooperation.

For the study of HCWs, a 1-page questionnaire was administered; HCWs were asked about exposure to persons with suspected Durba syndrome at work and at home, as well as any history of a compatible illness in the HCWs themselves. HCWs were also asked if they had ever entered the mines. Interviews were conducted in French.

After administration of the questionnaire, 5 mL of blood was obtained and stored out of the sunlight. At the end of the day, the serum and clot were separated, labeled, and stored in liquid nitrogen. Aliquots were sent on dry ice for analysis at both the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA, and the National Institute for Communicable Diseases in Johannesburg, South Africa.

Testing for Marburg-specific immunoglobulin (Ig) M and IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) was conducted with a technique analogous to that reported for detecting antibody to Ebola virus, except for the substitution of an antigen made from the Musoke strain of Marburg virus (30,31). The cut-off value for a positive ELISA result was set to 3 standard deviations from the mean control-adjusted optical density (OD) of 410 nm found on a panel of normal serum samples. This value generally corresponds to an OD of approximately 0.1 at a dilution of 1:100 for the IgM assay, and 0.2 at 1:400 for the IgG assay, which generally has a higher background. Positive and negative controls were included with each run and consisted of serum from African patients with and without a laboratory-confirmed history of MHF. Because the rarity of MHF has precluded rigorous field testing of the ELISA for Marburg antibody, all ELISA-positive serum samples were also examined by the immunofluorescent antibody assay (IFA), as previously described (32). The cut-off titer for a positive IFA result was 1:50. Serum samples were considered positive only if positive results were obtained by both ELISA and IFA. Study participants were directed to seek the serologic results (free of charge) 1 month after testing from the nurse in charge of the local health center. The nurse was provided the results along with details on how to interpret antibody status.

Questionnaires were created by using EpiInfo version 6.04 (CDC, Atlanta, GA). Data were initially recorded on the questionnaires by hand, then entered into EpiInfo 6.04, and finally imported into SPSS version 10.0 statistical software (SPSS, Chicago, IL) for further analysis. The chi-square test, Fisher exact test, and Student t test with Levene’s test for equality of variances were employed, where appropriate. Data found to be non-normally distributed were normalized by a log 10 transformation before statistical analysis. Statistical tests were two-sided, and significance was set at p < 0.05. Variables with significance values of p < 0.1 in the univariate analysis were examined in a multivariate model using forward stepwise maximum likelihood logistic regression.

A total of 912 participants were surveyed. Another seven persons were initially enrolled but did not stay to complete their questionnaires or to have a blood sample drawn. No further information is available regarding these seven or their reasons for withdrawal. Demographic data are presented in Table 1. Since the gold deposits in Durba attract young male miners from the surrounding region, most participants were men (65%), and 347 (38%) listed their occupation as miner, although only 281 (81%) of these 347 were currently working in the mines. Virtually all (99%) of the miners were male. Marburg-specific IgG antibodies were found in 15 (2%) of the 912 study participants. All were IgM antibody–negative. Thirteen (87%) of the 15 IgG antibody–positive participants were male miners. The other two were women who had never entered into the mines and whose profession was listed as “other” (most likely housewives).

All 13 of the antibody-positive miners were currently working: 10 (77%) at Goroumbwa, 2 (15%) at another mine in Durba, and 1 (8%) at an unspecified site. None reported river-diving for gold. Compared with those who were antibody-negative, antibody-positive miners tended to have worked fewer years at their present mine site but to have spent more time in the mines, although the differences were not statistically significant (Table 2). Antibody-positive miners were significantly younger than their antibody-negative counterparts (Table 1). This finding perhaps reflects the longer exposure time in the mines of younger miners (age <30 years) relative to older miners (mean + standard error of the mean [SEM] consecutive hours per week 17.7 + 1.2 vs. 13.7 + 1.3, respectively, p = 0.006; mean + SEM longest stint: 34.7 + 2.3 vs. 23.5 + 2.1, respectively, p < 0.001).

We examined associations between antibody to Marburg virus and various practices while working in the mines, as well as exposures related to sick persons in the home, healthcare services, and animals. In a univariate analysis, significant positive associations were found with having touched the corpse of someone who died from Durba syndrome, having had Durba syndrome oneself, and having received injections in the past year (Table 3). Touching the blood, feces, or urine of someone with Durba syndrome was of borderline statistical significance. Consumption of rodents was associated with a borderline significant protective effect, which was probably a spurious association. Of the four antibody-positive survey participants who said they had had Durba syndrome themselves, three dated the illness to the 5 months immediately before the study. The fourth, although uncertain, dated his illness to September 1998, 9 months before the investigation.

Receiving an injection as part of medical treatment was common in Durba: 505 (56%) of 907 of the participants in our cross-sectional village survey reported receiving an injection in the previous year, including 13 (87%) of the 15 antibody-positive participants (11 miners and both female nonminers). Overall, however, receiving injections was significantly more common among nonminers than miners (62 [368/596] vs. 47 [162/348], respectively, OR 1.9, 95% CI 1.4 to 2.4, p < 0.001) and among women than men (71% [221/312] vs. 49% [305/626], respectively, OR 2.6, 95% CI 1.9 to 3.4, p < 0.001).

In a multivariate model, the only variables that remained significantly associated with a positive Marburg antibody result were being a miner (OR 13.8, 95% CI 3.1 to 62.1) and having received injections (OR 7.4, 95% CI 1.6 to 33.2). Having previously had Durba syndrome was not added to the model, as it was not an independent risk factor for acquiring MHF. The associations between Marburg antibody, mining, and receiving injections remained essentially unaltered when men were looked at independently. The number of antibody-positive women (two) was too small to permit meaningful statistical analysis. However, both positive women were among the relatively few survey participants with extensive secondary contact in the household. Both reported having someone in the household sick with Durba syndrome, having contact with their body fluids, and participating in their burial, although only one of the two women reported direct contact with the corpse. In contrast, 4 (31%) of the 13 antibody-positive male miners reported any type of household exposure.

One hundred three HCWs were enrolled from 15 different health centers, including 73 (71%) nurses, 13 (13%) clerical or administrative staff, 10 (10%) midwives, 5 (5%) laboratory workers, and 2 (2%) doctors. These figures are thought to represent virtually all of the active HCWs in the two villages except those practicing traditional medicine. HCWs had a mean of 9 years (range 0–42) of experience. All were negative for both Marburg IgM and IgG antibodies, despite the fact that 67 (65%) reported caring for a patient with Durba syndrome, and 5 (5%) reported having had Durba syndrome themselves. Types of patient contact included administering injections (38%); cleaning up blood, vomitus, urine, or feces (28%); washing bed clothes (7%); washing corpses (6%); and receiving a needlestick injury (2%).