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    Figure 1.

    Likouala region in Republic of Congo outlined by the black box.

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    Figure 2.

    Eight locations of sample collection in Likouala region, Republic of Congo.

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    Figure 3.

    Optical density (OD) cut-off values (COVs) for orthopoxvirus IgG among patients at Talangai Hospital (black) compared with the Likouala population less than 25 years of age (gray).

  • 1

    Learned LA, Reynolds MG, Wassa DW, Li Y, Olson VA, Karem K, Stempora LL, Braden ZH, Kline R, Likos A, Libama F, Moudzeo H, Bolanda JD, Tarangonia P, Boumandoki P, Formenty P, Harvey JM, Damon IK, 2005. Extended interhuman transmission of monkeypox in a hospital community in the Republic of the Congo, 2003. Am J Trop Med Hyg 73 :428–434.

    • Search Google Scholar
    • Export Citation
  • 2

    Breman JG, Kalisa R, Steniowski MV, Zanotto E, Gromyko AI, Arita I, 1980. Human monkeypox, 1970–79. Bull World Health Organ 58 :165–182.

    • Search Google Scholar
    • Export Citation
  • 3

    Jezek Z, Nakano JH, Arita I, Mutombo M, Szczeniowski M, Dunn C, 1987. Serological survey for human monkeypox infections in a selected population in Zaire. J Trop Med Hyg 90 :31–38.

    • Search Google Scholar
    • Export Citation
  • 4

    Marennikova SS, Seluhina EM, Mal’eva NN, Cimiskjan KL, Macevic GR, 1972. Isolation and properties of the causal agent of a new variola-like disease (monkeypox) in man. Bull World Health Organ 46 :599–611.

    • Search Google Scholar
    • Export Citation
  • 5

    Lourie B, Bingham PG, Evans HH, Foster SO, Nakano JH, Hermann KL, 1972. Human infection with monkeypox virus: laboratory investigation of six cases in West Africa. Bull World Health Organ 46 :633–639.

    • Search Google Scholar
    • Export Citation
  • 6

    Khodkevich L, Widy-Wirski R, Arita I, Marennikova SS, Nakano JH, Meunier D, 1985. Orthopoxvirose simienne de l’omme en Republique Centrafricaine. Bull Soc Pathol Exot 78 :311–320.

    • Search Google Scholar
    • Export Citation
  • 7

    Damon IK, Roth CE, Chowdhary V, 2006. Discovery of monkeypox in Sudan. N Engl J Med 355 :962–963.

  • 8

    Centers for Disease Control and Prevention, 2003. Multistate outbreak of monkeypox–Illinois, Indiana, and Wisconsin, 2003. MMWR Morb Mortal Wkly Rep 52 :537–540.

    • Search Google Scholar
    • Export Citation
  • 9

    Huhn GD, Bauer AM, Yorita K, Graham MB, Sejvar J, Likos A, Damon IK, Reynolds MG, Kuehnert MJ, 2005. Clinical characteristics of human monkeypox, and risk factors for severe disease. Clin Infect Dis 41 :1742–1751.

    • Search Google Scholar
    • Export Citation
  • 10

    Nalca A, Rimoin AW, Bavari S, Whitehouse CA, 2005. Reemergence of monkeypox: prevalence, diagnostics, and countermeasures. Clin Infect Dis 41 :1765–1771.

    • Search Google Scholar
    • Export Citation
  • 11

    Jezek Z, Szczeniowski M, Paluku KM, Mutombo M, Grab B, 1988. Human monkeypox: confusion with chickenpox. Acta Trop 45 :297–307.

  • 12

    Reynolds MG, Yorita KL, Kuehnert MJ, Davidson WB, Huhn GD, Holman RC, Damon IK, 2006. Clinical manifestations of human monkeypox influenced by route of infection. J Infect Dis 194 :773–780.

    • Search Google Scholar
    • Export Citation
  • 13

    Likos A, Sammons SA, Olson VA, Frace AM, Li Y, Olsen-Rasmussen M, Davidson W, Galloway R, Khristova ML, Reynolds MG, Zhao H, Carroll DS, Curns A, Formenty P, Esposito JJ, Regnery RL, Damon IK, 2005. A tale of two clades: monkeypox viruses. J Gen Virol 86 :2661–2672.

    • Search Google Scholar
    • Export Citation
  • 14

    World Health Organization, 1988. Human Monkeypox and Other Poxvirus Infections of Man. Volume 29. Geneva: World Health Organization.

  • 15

    Jezek Z, Grab B, Szczeniowski M, Paluku KM, Mutombo M, 1988. Clinico-epidemiological features of monkeypox patients with an animal or human source of infection. Bull World Health Organ 66 :459–464.

    • Search Google Scholar
    • Export Citation
  • 16

    Rimoin AW, Kisalu N, Kebela-Ilunga B, Mukaba T, Wright LL, Formenty P, Wolfe ND, Shongo RL, Tshioko F, Okitolonda E, Muyembe JJ, Ryder RW, Meyer H, 2007. Endemic human monkeypox, Democratic Republic of Congo, 2001–2004. Emerg Infect Dis 13 :934–937.

    • Search Google Scholar
    • Export Citation
  • 17

    Karem KL, Reynolds M, Braden Z, Lou G, Bernard N, Patton J, Damon IK, 2005. Characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak. Clin Diagn Lab Immunol 12 :867–872.

    • Search Google Scholar
    • Export Citation
  • 18

    CIA, The World Fact Book. Accessed August 10, 2006. Available from https://www.cia.gov/cia/publications/factbook/geos/cf.html

  • 19

    Talani P, Maniane-Nanga J, Konongo JD, Gromyko AI, Yala F, 1999. Prevalence des anticorps specifiques du monkeypox au Congo-Brazzaville. Med Afr Noire 46 :421–423.

    • Search Google Scholar
    • Export Citation
  • 20

    Downie AW, Mc Carthy K, 1958. The antibody response in man following infection with viruses of the pox group. III. Antibody response in smallpox. J Hyg (Lond) 56 :479–487.

    • Search Google Scholar
    • Export Citation
  • 21

    Chastel C, Charmot G, 2004. Bacterial and viral epidemics of zoonotic origin; the role of hunting and cutting up wild animals. Bull Soc Pathol Exot 97 :207–212.

    • Search Google Scholar
    • Export Citation
  • 22

    Ladnyj ID, Ziegler P, Kima E, 1972. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ 46 :593–597.

    • Search Google Scholar
    • Export Citation
  • 23

    Kemble SK, Davis JC, Nalugwa T, Njama-Meya D, Hopkins H, Dorsey G, Staedke SG, 2006. Prevention and treatment strategies used for the community management of childhood fever in Kampala, Uganda. Am J Trop Med Hyg 74 :999–1007.

    • Search Google Scholar
    • Export Citation
  • 24

    Yamamoto K, Chomel BB, Lowenstine LJ, Kikuchi Y, Phillips LG, Barr BC, Swift PK, Jones KR, Riley SP, Kasten RW, Foley JE, Pedersen NC, 1998. Bartonella henselae antibody prevalence in free-ranging and captive wild felids from California. J Wildl Dis 34 :56–63.

    • Search Google Scholar
    • Export Citation
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Prevalence of Antibodies against Orthopoxviruses among Residents of Likouala Region, Republic of Congo: Evidence for Monkeypox Virus Exposure

Edith R. LedermanPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Mary G. ReynoldsPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Kevin KaremPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Zachary BradenPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Lynne A. Learned-OrozcoPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Demole Wassa-WassaPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Omba MoundeliPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Christine HughesPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Joseph HarveyPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Russell RegneryPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Jean-Vivien MombouliPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Inger K. DamonPoxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; School of Medicine, State University of New York, Buffalo, New York; Ministry of Health, Pointe Noire, Republic of Congo; District Health Office, Impfondo, Republic of Congo; Pioneer Christian Hospital, Impfondo, Republic of Congo; National Laboratory, Brazzaville, Republic of Congo

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Monkeypox virus is a zoonotic orthopoxvirus (OPX) of west and central sub-Saharan Africa. We conducted a cross-sectional serosurvey in Likouala region, Republic of Congo to assess exposure to OPX. Whole blood was collected using Nobuto blood filter strips (NBFS). Titers of IgM and IgG to OPX were assessed using an enzyme-linked immunosorbent assay. Demographic and clinical characteristics were compared with serostatus using the chi-square test or Fisher’s exact test. Multivariate logistic regression was performed to evaluate factors for independent association with serostatus. A total of 994 specimens were analyzed; the overall seroprevalence for OPX IgM was 1.7%. Age < 25 years reduced the likelihood of OPX exposure, and persons living in Ngangania village had independently higher odds (odds ratio = 33.5, 95% confidence interval = 7.2–166). Blood collection for serosurveys using NBFS is feasible and practical. Adult activities such as hunting and carcass preparation may play an important role in exposure to Monkeypox virus.

INTRODUCTION

Monkeypox virus is a zoonotic orthopoxvirus (OPX), the primary animal host, or hosts, of which remains unknown. Human monkeypox cases have been reported sporadically in west Africa and more often in the Congo Basin,16 and most recently, Sudan.7 The sole report of human monkeypox in the Western hemisphere occurred in association with imported African rodents.8,9 Classic descriptions of human monkeypox denote an illness that resembles discrete, ordinary smallpox (caused by Variola virus), but human monkeypox can be distinguished by the presence of prominent lymphadenopathy, which occurs in 70–82% of cases.911 The route of infection (percutaneous versus inhalational),12 strain type (Congo Basin versus West African),13 and comorbidities may affect the severity of illness. Case-fatality rate reports vary from 0% to 10%.9,14 Human-to-human transmission of Monkeypox virus is presumed to be inefficient relative to variola,15 but incidents of extended chains of transmission have been documented.1 Many aspects of monkeypox illness have yet to be described, including exposure rates in the general population of virus-endemic areas, but research is ongoing in the Democratic Republic of Congo where human cases are routinely evaluated.16 Such information can be useful in identifying locales of interest for future ecologic studies, documenting the extent of sub-clinical disease, and evaluating subgroups of the population who have the greatest life-time risk of contracting an infection with Monkeypox virus.

In 2003, an outbreak of community-acquired monkeypox infection with hospital-associated spread was documented in Likouala region, Republic of Congo1 (Figure 1). The index case was a patient from the northern town of Dongou. Since the outbreak, anecdotal reports of monkeypox disease from other regions of Likouala region have been recounted by local health workers. In conjunction with a Congolese Ministry of Health community health survey, we conducted a serosurvey evaluating antibodies to OPX, both IgG (possible surrogate for monkeypox exposure in vaccinia- and variola-naive individuals), and IgM (indicative of recent non-vaccinia OPX exposure in smallpox vaccinated and unvaccinated individuals).17 The purpose of this survey was to assess the frequency and distribution of Monkeypox virus exposure in residents of Likouala region.

MATERIALS AND METHODS

Location and population.

This serosurvey was conducted in eight locations in Likouala region in northeastern Republic of Congo: Impfondo (the regional capital), Dongou (district seat), and the villages of Botala, Botanga, Bokota, Ngangania, Matoko, and Bonzali (Bondzale) (Figure 2). The villages were adjacent to inundated rain forest, located either along a paved road that paralleled the Ubangi River or along the road southeast toward the protected Lac Tele reserve. Housing consisted of single-unit dwellings constructed mainly of mud brick with thatched or tin roofs, in addition to concrete structures in the two cities of Impfondo and Dongou. Municipal water and electricity existed only sporadically in Impfondo. Two hospitals provided care for the region: Impfondo District Hospital and the Pioneer Christian Hospital (also in Impfondo); health clinics and health posts provided routine on-site care in the villages.

Approximately 84,500 residents live in Likouala region, which is further subdivided into 7 districts. The catchment area included eight communities in three of these districts (Impfondo, Dongou, and Epena) with approximately 40,650 residents. The population consists of subsistence farmers (cassava, corn), small business owners, and hunters. Several ethnic groups co-existed including the Bantu (ethnic majority) and the Aka (pygmy).

Serosurvey and specimen collection.

This serosurvey was conducted in concert with a Ministry of Health hemoglobin survey in seven of the eight locations and was de facto a convenience sample representing communities of different sizes located on transportation axes between Impfondo and one of two outlying health posts (Epena and Dongou). Only persons voluntarily participating in the Ministry of Health-sponsored hemoglobin survey were included in this investigation (i.e., no structured sampling was performed). The specimens from the eighth location (Impfondo) were collected from routine clinical specimen remainders at the Pioneer Christian Hospital. Additional specimens were collected in a likewise fashion (i.e., convenience sample) at the Talangai Hospital in Brazzaville (the capital city of Republic of Congo) for serologic comparison only (urban versus rural/ virus-endemic area). Non-identifying data, such as age range (> 25 years of age or ≤ 25 years of age to distinguish those who may have been vaccinated during the smallpox eradication campaign) and sex, as well as illness information (e.g., fever, rash) in the preceding six months were collected. After the whole blood specimen was collected for the hemoglobin analysis by an aseptically performed finger stick, a Nobuto blood filter strip (NBFS) (Advantec MFS, Inc., Dublin, CA) was used to collect the remaining whole blood from the puncture site. This was used for analysis of antibodies to OPX. Because of variability in the amount of blood remaining on the finger, the amount of blood collected ranged from 20 μL to 100 μL. The NBFSs were marked with a number that linked the data to the strip, air-dried, and placed in a plastic zip-lock bag with silica desiccant for shipment to the Centers for Disease Control and Prevention (CDC) poxvirus laboratory for serologic testing. The methods and data collection for this investigation were reviewed and approved by the National Centers for Infectious Diseases and Division of Viral and Rickettsial Diseases (CDC) Institutional Review Board coordinating officials and were determined to be within the scope of infectious disease surveillance and thereby exempt from Institutional Review Board review.

Laboratory techniques.

Upon receipt at the poxvirus laboratory, the NBFSs were assessed for volume of blood collected and the volume of elution buffer, phosphate-buffered saline (PBS), was determined based on the fraction of the NBFS covered with blood (as per manufacturer’s protocol). An appropriate, relative volume of PBS was then added to the microfuge tube to achieve a serum dilution of 1:40 based on manufacturer’s instructions, vortexed briefly, and the mixture was refrigerated overnight. Samples were tested by enzyme-linked immunosorbent assay (ELISA) for IgM and IgG antibodies to OPX as previously described.17 Briefly, the IgG ELISA is an indirect assay using purified vaccinia virus–coated plates, extracted NFBS samples (1:40 dilution), and goat anti-human IgG horseradish peroxidase conjugate. The IgM ELISA is an indirect capture assay using goat anti-human IgM coated plates, extracted NFBS samples (1:40 dilution), purified Vaccinia virus, anti-Variola virus hyperimmune mouse polyclonal ascitic fluid, and goat anti-mouse IgG horseradish peroxidase conjugate. Known anti-OPX positive and negative human whole blood samples from African sources were wicked onto NBFS and were used as controls for this analysis. Assay cut-off values (COVs) were defined as the mean plus three standard deviations of five known negative specimens. Results are reported as the optical density (OD) minus the COV, where any value above zero may be considered positive and any below zero will be considered negative. For additional rigor, a secondary designation of highly positive was assigned to any specimen that exceeded the highest positive OD minus COV in the comparator population (i.e., Talangai Hospital in Brazzaville). As previously described, the anti-OPX IgG assay has a sensitivity of 100% and a specificity of 88.5%; the IgM anti-OPX assay has a sensitivity of 92% and a specificity of 100%.17 Although both assays are specific for the genus Orthopoxvirus, they cannot differentiate between antibodies elicited by different orthopoxvirus species within this genus.

Data analysis.

Data was analyzed using EpiInfo 2000 software (CDC, Atlanta, GA). Means were calculated for continuous variables. Univariate analysis was performed for categorical variables using the chi-square test or Fisher’s exact test, as appropriate. Linear regression was used to analyze continuous variables for association. A logistic regression model was designed using all variables statistically significant in the univariate analysis to establish the variables that were independently significant (P < 0.05).

RESULTS

Whole blood specimens from 994 Likouala residents were collected during this serosurvey, which translates to a coverage of from 0.4% to 76.0% of the population in any given area (Table 1). In three villages, at least 50% of the entire population was sampled. We estimate an overall capture of 2.4% of our catchment area in the Likouala region. The overall amplitude of seropositive specimens was higher in the Likouala specimens when compared with the Brazzaville population (Figure 3).

Nearly three-fourths of the survey population was younger than 25 years of age (the primary objective of the concurrent Ministry of Health hemoglobin survey was to estimate anemia in children). However, because 46% of the Congolese population is estimated to be less than 15 years of age, this may still be representative of the general population.18 Because of the rural location, we captured a disproportionate number of Aka (pygmy ethnic group) residents (8% of the survey population); this ethnic group comprises less than 1% of the Congolese population.

Overall, 56.9% of those surveyed in Likouala had IgG antibodies to OPX, and 1.7% had IgM antibodies to OPX (Table 2). Taking no other factors into account (i.e., without controlling for additional variables) OPX IgG seroprevalence was significantly higher in those more than 24 years of age, in women, in those with a fever in the past 6 months, and in those living in Impfondo. OPX IgM seroprevalence was significantly higher in those more than 24 years of age, in those of the Aka ethnic group, and in Ngangania village. Positive serostatus for IgG antibody to OPX varied between villages in individuals ≤ 25 years of age but mirrored the seroprevalence of individuals > 25 years of age; the greatest exposure occurred in the axis to the south and west of Impfondo (Impfondo 76%, Matoko 53%, Botala 51%, Ngangania 45%). Likewise, IgM antibody to OPX ranged from 0% to 9%.

Multivariate analysis for individuals positive for IgG antibodies to OPX by village was performed for all significant variables found in the univariate analysis (Table 3). Age less than 25 years or being a resident of a northern route village (Dongou, Bondzale, Botanga) was protective; being a resident of Impfondo (regional capital) or having a fever in the last six months was independently associated with having IgG antibodies to OPX. Table 4 shows the results of the multivariate analysis for individuals with OPX IgM seropositivity. Being a resident of Ngangania village was the only independent risk factor associated with positive IgM serostatus (odds ratio [OR] = 35.3, 95% confidence interval [CI] = 7.5– 166) whereas being less than 25 years of age or living in Impfondo city appeared to independently protective. The finding of independent significance for risk related to living in Ngangania village held true even when the more stringent highly positive category was used to designate subjects positive for IgG antibody to OPX (highly positive; OR = 44.7, 95% CI = 3.2–627). The highly positive classification introduces an additional degree of stringency because this category includes only OD-COV values that are above the level of potential background observed in a Congolese urban setting (Talangi Hospital, Brazzaville).

An incidence estimate for primary non-vaccine– derived OPX infection may be extrapolated given the following assumptions: 1) having significant levels of IgM antibodies to OPX is consistent with having had an infection with Monkey-pox virus in the previous year, regardless of prior smallpox vaccination history17; 2) the age distribution throughout Likouala region is constant and is 62% for those ≤ 25 years of age and 48% for those > 25 years of age (estimates per Republic of Congo Ministry of Health); 3) risk of disease exposure is uniform throughout the region; 4) repeated exposures to Monkeypox virus will not elicit an IgM response. Therefore, with a total population of 84,500, 52,390 individuals would be < 25 years of age (i.e., never vaccinated for or exposed to Variola virus). On the basis of population demographics and IgM seroprevalence in this age group (1.7%), there are potentially 890 primary Monkeypox virus infections in persons ≤ 25 years of age per year in Likouala region (1,691 infections/100,000 persons). By the same method, for those individuals more than 25 years of age, we would expect 1,825 potential primary infections per year in Likouala region (5,658 infections/100,000 population).

DISCUSSION

We conducted a serosurvey of antibodies to OPX among residents in three health districts of Likouala region, Republic of Congo, the site of a monkeypox outbreak in 2003.1 Several decades ago, Jezek and others found an overall seroprevalence of 19% for total antibodies to OPX detected using a hemagglutinin inhibition assay (HIA) and monkeypox-specific seroprevalence of 0.8% (0.7% for those less than 30 years of age by radioimmuneassay adsorption) in serum of residents of Kole, Democratic Republic of Congo.3 Similarly, Talani and others found a seroprevalence of antibody to OPX of 16% in residents of Pool and Sangha provinces of Republic of Congo.19 This is in contrast to our study, which found the overall anti-OPX IgG seroprevalence to be 56.9%. Given that the estimated smallpox vaccine coverage in this region was 50%, a fair proportion of adult seroreactivity could be attributed to exposure to smallpox vaccine. A greater predominance of younger individuals (90% less than 30 years of age in the Kole study and 100% less than 15 years of age in the Pool/Sangha study) may account for the differences in sero-prevalence between our study and previous studies because most in these populations would not have been vaccinated. Furthermore, antibodies detected by HIA to orthopoxviruses may be shorter lived than those detected by ELISA.20 Our findings may represent a higher sensitivity with our assay or higher rates of infection with an orthopoxvirus (presumably Monkeypox virus). There are several possible explanations that could account for the latter hypothesis. For example, higher levels of Monkeypox virus exposure may be the result of changing patterns of human interaction with sylvan animals (i.e., larger human populations at the margins of densely forested areas). Conversely, waning herd immunity caused by eradication of variola, and recruitment of virus-naive persons into the population after cessation of the smallpox vaccination program over 20 years ago could have led to increased population-level susceptibility to all orthopoxviruses, including Monkeypox virus.

Several significant associations with OPX seroprevalence were apparent. Older participants were more likely to have detectable IgG and IgM antibodies to OPX than their younger counterparts. As indicated above, this differential of IgG antibody to OPX can in part be accounted for by previous exposure to vaccinia and/or variola viruses; some contribution may also be caused by Monkeypox. The presence of IgM antibody to OPX is indicative of recent orthopoxvirus exposure. Its association with the older age group may be linked to activities more likely performed by adults,21 such as hunting and food preparation. Hunting as well as handling animal carcasses in the village setting have been postulated to be a source of Monkeypox virus exposure in previous studies.15,22

Although female sex was significantly associated with seropositivity for IgG antibodies to OPX by univariate analysis, we did not find sex to be an independent risk factor for exposure to OPX; this is in contrast to a previous study that found men to be more commonly infected with Monkeypox virus.15 Females had greater odds of reporting fever in the previous six months (71% of females versus 65% of males report fever) and living in Impfondo city (60% of Impfondo subjects were female), thus leading to an apparent association between female sex and IgG antibodies to OPX by univariate analysis.

Having had a fever in the six months prior to the survey was associated with having IgG antibodies to OPX but not IgM antibodies to OPX. However, the rate of febrile illness in a rural sub-Saharan African population is expected to be high,23 which increases the possibility for a non-specific association to be detected. In addition, one of the two locations with fever as a significant finding was Impfondo and NBFS were taken from patient clinical specimens; concurrent illness could easily have led to fever as a significant finding. The other location (Dongou) had a survey population with a high proportion of Democratic Republic of Congo refugees (24%); higher concomitant illness in this population is also expected given the increased vulnerability of displaced persons, and may account for the higher prevalence of fever. Associations with generic rash or vesiculopustular rash were not found with either IgG or IgM antibodies to OPX. We were unable to determine whether poor subject recall, subclinical infections, or other factors might have contributed to these findings, but the potential role of each deserves additional future attention.

The only location associated with a higher seroprevalence of IgM antibody to OPX was Ngangania. Forty-two percent of individuals surveyed in this village were of Aka pygmy ethnicity (an ethnic group of traditional hunter-gatherers) by Ministry of Health personnel. However, Aka ethnicity was not significantly associated with seropositivity for IgM antibodies to OPX in Ngangania (12.8% versus 6.3%; P = 0.2). The suggestion of a recent focus of infection in Ngangania village argues that this village and its associated garden plots could provide a suitable locale for ecologic studies to identify the animal host(s) of Monkeypox virus.

This survey used NBFSs for collection of specimens. These strips are lightweight, do not require refrigeration for storage, are relatively inexpensive (pennies a strip), and are calibrated to allow for standardized quantification regardless of the amount of blood collected. Although assays performed with NBFSs should be validated against routinely collected serum specimens (i.e., venipuncture and collection in serum separator glass tubes), NBFSs offer a practical alternative for blood collection in rural areas and have been used in serosurveys of other infectious diseases.24

There are several limitations to our survey. We used a generic OPX assay as a surrogate marker for exposure to Monkeypox virus in a region where monkeypox has been observed. Given cross-reactivity with Vaccinia virus and Variola virus, this assay would not be specific for Monkeypox virus exposure in individuals who were alive during the smallpox eradication campaign. Therefore, positive serostatus for IgG antibodies to OPX in this group could be caused by natural infection with Variola virus, vaccination with Vaccinia virus, and/or exposure to Monkeypox virus. However in 2006, for those less than 25 years of age, barring exposure to another currently unrecognized OPX, the assay for IgG antibodies to OPX is hypothesized to be indicative of previous Monkeypox virus exposure. Furthermore, 80% of persons positive for IgM antibodies to OPX who have been exposed to Monkeypox virus have elevated titers for ≤ 1 year (Karem K, unpublished data).

A second limitation of this survey stems from using a convenience sample (albeit one that represented a thorough sampling of potentially affected communities) rather than a population-based sample to generate incidence and prevalence estimates. This survey will however provide a solid foundation to build future epidemiologic studies that will attempt to gather more detailed information regarding risk factors for exposure to Monkeypox virus (e.g., hunting, rodent exposures). Furthermore, because this survey was limited to residents of northeastern Republic of Congo, the results may not be generalizable to all areas endemic for Monkeypox virus.

Another noteworthy limitation involves the blood collection method (i.e., NBFS). Although NBFSs are inexpensive, practical (i.e., do not require a cold chain), and easy to use, they are limited in the volume of blood they can hold (maximum = 100 μL). However, we did have sufficient NBFS volume to complete assays for IgM and IgG antibodies to OPX for 98.2% of specimens collected. However, little sample, if any, remained for additional testing.

In summary, our survey provides substantive evidence to support observations of ongoing exposure to, and infection with, Monkeypox virus in residents of Likouala region, Republic of Congo, including a likely focus of recent (i.e., the past year) transmission in and around the village of Ngangania.

Table 1

Monkeypox serosurvey population coverage by location, Likouala Region, Republic of Congo

LocationPopulation estimate*No. individuals sampled% coverage by serosurvey
* All population estimates exclude refugees; Source Global Gazzetter2.1, Falling Rain Genomics Inc, 1996 and projection from 2001 census.
Dongou13,900500.4
Bondzale15011073.3
Botanga1007676.0
Bokata502958.0
Impfondo24,5002871.2
Ngangania35011132.3
Botala60012020.0
Matoko1,00021121.1
Total40,6509942.4
Table 2

Univariate analysis of demographic, clinical, and geographic variables in comparison with IgM and IgG against orthopoxviruses for residents of Likouala Region, Republic of Congo*

Variable% Total survey population (n = 994)% IgG positive (n = 566)P% IgM positive (n = 17)P
* 978 specimens were analyzed for IgG and 976 specimens were analyzed for IgM (equivocal considered negative). NS = not significant; VP = vesiculopustular.
† Sex was unknown for 4 persons.
‡ Ethnicity was unknown for 288 persons.
§ Country of origin was unknown for 172 persons.
¶ Fever status was unknown for 19 persons.
# VP rash status was unknown for 55 persons.
** % in those > 25 years of age.
Total survey56.91.7
Age ≤ 25 years72.049.11.1
Age > 25 years28.076.7< 0.00013.30.025
Female†54.760.41.3
Male44.051.60.0062.1NS
Ethnic Aka‡8.252.47.3
Not ethnic Aka62.845.3NS1.50.005
Refugee§4.768.12.1
Not a refugee78.056.4NS1.8NS
Fever in past 6 months (Yes)¶67.162.51.8
No fever in the past 6 months31.242.8< 0.00011.3NS
VP rash in the past 6 months#1.850.00.0
No VP rash in the past 6 months92.655.9NS1.8NS
Location
Dongou, n = 505.028.6 (3.2)**< 0.00010.0 (0.0)**NS
Bondzale, n = 11011.143.1 (25.0)0.0020.9 (0.0)NS
Botanga, n = 767.623.7 (9.1)< 0.00011.3 (0.0)NS
Bokata, n = 292.958.6 (33.3)NS3.4 (0.0)NS
Impfondo, n = 28728.982.9 (75.2)< 0.00010.7 (0.0)NS
Ngangania, n = 11111.150.9 (44.3)NS9.0 (6.8)< 0.0001
Botala, n = 12012.150.4 (49.5)NS0.0 (0.0)NS
Matoko, n = 21121.253.4 (51.2)NS0.9 (1.0)NS
Table 3

Multivariate analysis and odds ratios (95% confidence intervals) by location of specimen collection for residents of Likouala region, Republic of Congo, with IgG against orthopoxviruses for variables of significance in univariate analysis*

VariableDongouBondzaleBotangaBokataImpfondoNganganiaBotalaMatoko
* NS = not significant.
Age < 25 years0.29 (0.21–0.40)0.29 (0.21–0.41)0.26 (0.18–0.37)0.29 (0.21–0.41)0.37 (0.26–0.52)0.31 (0.22–0.43)0.30 (0.22–0.42)0.29 (0.21–0.41)
FemaleNSNSNSNSNSNSNSNS
Village0.28 (0.14–0.55)0.55 (0.36–0.86)0.19 (0.10–0.34)NS4.1 (2.8–5.9)NSNSNS
Fever2.1 (1.6–2.8)2.1 (1.6–2.8)1.9 (1.4–2.6)2.2 (1.7–3.0)1.5 (1.1–2.0)2.3 (1.7–3.0)2.3 (1.7–3.0)2.2 (1.7–3.0)
Table 4

Multivariate analysis and odds ratios (95% confidence intervals) by location of specimen collection for residents of Likouala region, Republic of Congo, with IgM against orthopoxviruses for variables of significance in univariate analysis*

VariableDongouBondzaleBotangaBokataImpfondoNganganiaBotalaMatoko
* NA = not available; NS = not significant.
Age < 25years0.23 (0.07– 0.8)0.20 (0.06– 0.72)0.23 (0.06– 0.8)0.22 (0.06– 0.81)0.28 (0.10– 0.77)0.22 (0.06– 0.78)0.27 (0.08– 0.9)0.24 (0.06– 0.9)
Aka13.9 (2.8– 70)17.7 (3.5– 88.2)14.8 (3.0– 73.2)15.7 (3.2– 77.8)NANS13.5 (2.7– 68.3)15.5 (3.0– 80)
VillageNSNSNSNS0.12 (0.02– 0.9)35.3 (7.5– 166)NSNS
Figure 1.
Figure 1.

Likouala region in Republic of Congo outlined by the black box.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1150

Figure 2.
Figure 2.

Eight locations of sample collection in Likouala region, Republic of Congo.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1150

Figure 3.
Figure 3.

Optical density (OD) cut-off values (COVs) for orthopoxvirus IgG among patients at Talangai Hospital (black) compared with the Likouala population less than 25 years of age (gray).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1150

*

Address correspondence to Edith R. Lederman, Naval Medical Center, 34800 Bob Wilson Drive, San Diego, CA 92134. E-mail: Edith.Lederman@med.navy.mil

Authors’ addresses: Edith R. Lederman, Naval Medical Center, 34800 Bob Wilson Drive, San Diego, CA 92134, E-mail: Edith.Lederman@med.navy.mil. Mary G. Reynolds, Kevin Karem, Zachary Braden, Christine Hughes, Russell Regnery, and Inger K. Damon, Poxvirus Program, Poxvirus and Rabies Branch, Division for Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop G-43. Atlanta, GA 30333. Lynne A. Learned-Oroczo, 603 Oakleigh Place, Brandon, MS 39047. Demole Wassa-Wassa, Omba Moundeli, and Jean-Vivien Mombouli, Department of Microbiology, Marien Ngouabi University, BP 69, Brazzaville, Republic of Congo. Joseph Harvey, Mission G.O. Congo, BP 10, Impfondo (par Brazzaville), Republic of Congo.

Acknowledgments: This data was presented in part at the 55th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, Georgia, November 2006.

Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention, the United States Navy or the U.S. Department of Defense.

REFERENCES

  • 1

    Learned LA, Reynolds MG, Wassa DW, Li Y, Olson VA, Karem K, Stempora LL, Braden ZH, Kline R, Likos A, Libama F, Moudzeo H, Bolanda JD, Tarangonia P, Boumandoki P, Formenty P, Harvey JM, Damon IK, 2005. Extended interhuman transmission of monkeypox in a hospital community in the Republic of the Congo, 2003. Am J Trop Med Hyg 73 :428–434.

    • Search Google Scholar
    • Export Citation
  • 2

    Breman JG, Kalisa R, Steniowski MV, Zanotto E, Gromyko AI, Arita I, 1980. Human monkeypox, 1970–79. Bull World Health Organ 58 :165–182.

    • Search Google Scholar
    • Export Citation
  • 3

    Jezek Z, Nakano JH, Arita I, Mutombo M, Szczeniowski M, Dunn C, 1987. Serological survey for human monkeypox infections in a selected population in Zaire. J Trop Med Hyg 90 :31–38.

    • Search Google Scholar
    • Export Citation
  • 4

    Marennikova SS, Seluhina EM, Mal’eva NN, Cimiskjan KL, Macevic GR, 1972. Isolation and properties of the causal agent of a new variola-like disease (monkeypox) in man. Bull World Health Organ 46 :599–611.

    • Search Google Scholar
    • Export Citation
  • 5

    Lourie B, Bingham PG, Evans HH, Foster SO, Nakano JH, Hermann KL, 1972. Human infection with monkeypox virus: laboratory investigation of six cases in West Africa. Bull World Health Organ 46 :633–639.

    • Search Google Scholar
    • Export Citation
  • 6

    Khodkevich L, Widy-Wirski R, Arita I, Marennikova SS, Nakano JH, Meunier D, 1985. Orthopoxvirose simienne de l’omme en Republique Centrafricaine. Bull Soc Pathol Exot 78 :311–320.

    • Search Google Scholar
    • Export Citation
  • 7

    Damon IK, Roth CE, Chowdhary V, 2006. Discovery of monkeypox in Sudan. N Engl J Med 355 :962–963.

  • 8

    Centers for Disease Control and Prevention, 2003. Multistate outbreak of monkeypox–Illinois, Indiana, and Wisconsin, 2003. MMWR Morb Mortal Wkly Rep 52 :537–540.

    • Search Google Scholar
    • Export Citation
  • 9

    Huhn GD, Bauer AM, Yorita K, Graham MB, Sejvar J, Likos A, Damon IK, Reynolds MG, Kuehnert MJ, 2005. Clinical characteristics of human monkeypox, and risk factors for severe disease. Clin Infect Dis 41 :1742–1751.

    • Search Google Scholar
    • Export Citation
  • 10

    Nalca A, Rimoin AW, Bavari S, Whitehouse CA, 2005. Reemergence of monkeypox: prevalence, diagnostics, and countermeasures. Clin Infect Dis 41 :1765–1771.

    • Search Google Scholar
    • Export Citation
  • 11

    Jezek Z, Szczeniowski M, Paluku KM, Mutombo M, Grab B, 1988. Human monkeypox: confusion with chickenpox. Acta Trop 45 :297–307.

  • 12

    Reynolds MG, Yorita KL, Kuehnert MJ, Davidson WB, Huhn GD, Holman RC, Damon IK, 2006. Clinical manifestations of human monkeypox influenced by route of infection. J Infect Dis 194 :773–780.

    • Search Google Scholar
    • Export Citation
  • 13

    Likos A, Sammons SA, Olson VA, Frace AM, Li Y, Olsen-Rasmussen M, Davidson W, Galloway R, Khristova ML, Reynolds MG, Zhao H, Carroll DS, Curns A, Formenty P, Esposito JJ, Regnery RL, Damon IK, 2005. A tale of two clades: monkeypox viruses. J Gen Virol 86 :2661–2672.

    • Search Google Scholar
    • Export Citation
  • 14

    World Health Organization, 1988. Human Monkeypox and Other Poxvirus Infections of Man. Volume 29. Geneva: World Health Organization.

  • 15

    Jezek Z, Grab B, Szczeniowski M, Paluku KM, Mutombo M, 1988. Clinico-epidemiological features of monkeypox patients with an animal or human source of infection. Bull World Health Organ 66 :459–464.

    • Search Google Scholar
    • Export Citation
  • 16

    Rimoin AW, Kisalu N, Kebela-Ilunga B, Mukaba T, Wright LL, Formenty P, Wolfe ND, Shongo RL, Tshioko F, Okitolonda E, Muyembe JJ, Ryder RW, Meyer H, 2007. Endemic human monkeypox, Democratic Republic of Congo, 2001–2004. Emerg Infect Dis 13 :934–937.

    • Search Google Scholar
    • Export Citation
  • 17

    Karem KL, Reynolds M, Braden Z, Lou G, Bernard N, Patton J, Damon IK, 2005. Characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak. Clin Diagn Lab Immunol 12 :867–872.

    • Search Google Scholar
    • Export Citation
  • 18

    CIA, The World Fact Book. Accessed August 10, 2006. Available from https://www.cia.gov/cia/publications/factbook/geos/cf.html

  • 19

    Talani P, Maniane-Nanga J, Konongo JD, Gromyko AI, Yala F, 1999. Prevalence des anticorps specifiques du monkeypox au Congo-Brazzaville. Med Afr Noire 46 :421–423.

    • Search Google Scholar
    • Export Citation
  • 20

    Downie AW, Mc Carthy K, 1958. The antibody response in man following infection with viruses of the pox group. III. Antibody response in smallpox. J Hyg (Lond) 56 :479–487.

    • Search Google Scholar
    • Export Citation
  • 21

    Chastel C, Charmot G, 2004. Bacterial and viral epidemics of zoonotic origin; the role of hunting and cutting up wild animals. Bull Soc Pathol Exot 97 :207–212.

    • Search Google Scholar
    • Export Citation
  • 22

    Ladnyj ID, Ziegler P, Kima E, 1972. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ 46 :593–597.

    • Search Google Scholar
    • Export Citation
  • 23

    Kemble SK, Davis JC, Nalugwa T, Njama-Meya D, Hopkins H, Dorsey G, Staedke SG, 2006. Prevention and treatment strategies used for the community management of childhood fever in Kampala, Uganda. Am J Trop Med Hyg 74 :999–1007.

    • Search Google Scholar
    • Export Citation
  • 24

    Yamamoto K, Chomel BB, Lowenstine LJ, Kikuchi Y, Phillips LG, Barr BC, Swift PK, Jones KR, Riley SP, Kasten RW, Foley JE, Pedersen NC, 1998. Bartonella henselae antibody prevalence in free-ranging and captive wild felids from California. J Wildl Dis 34 :56–63.

    • Search Google Scholar
    • Export Citation
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