Emerg Infect DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention17553242279285006-154010.3201/eid1306.061540DispatchEndemic Human Monkeypox, Democratic Republic of Congo, 2001–2004Human Monkeypox, Democratic Republic of CongoRimoinAnne W.*KisaluNevilleKebela-IlungaBenoitMukabaThibautWrightLinda L.§FormentyPierreWolfeNathan D.#ShongoRobert LoshimaTshiokoFlorimond**OkitolondaEmile††MuyembeJean-JacquesRyderRobert W.‡‡MeyerHermann§§University of California, Los Angeles, California, USANational Institute of Biomedical Research, Kinshasa, Democratic Republic of CongoMinistry of Health, Kinshasa, Democratic Republic of CongoNational Institutes of Health, Bethesda, Maryland, USAWorld Health Organization, Geneva, SwitzerlandJohns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAWorld Health Organization, Kinshasa, Democratic Republic of CongoKinshasa School of Public Health, Kinshasa, Democratic Republic of CongoUniversity of North Carolina, Chapel Hill, North Carolina, USABundeswehr Institute of Microbiology, Munich, GermanyAddress for correspondence: Hermann Meyer, Bundeswehr Institute of Microbiology, Neuherbergstr. 11, D-80937 Munich, Germany; email: hermann1meyer@bundeswehr.org62007136934937

By analyzing vesicle fluids and crusted scabs from 136 persons with suspected monkeypox, we identified 51 cases of monkeypox by PCR, sequenced the hemagglutinin gene, and confirmed 94% of cases by virus culture. PCR demonstrated chickenpox in 61 patients. Coinfection with both viruses was found in 1 additional patient.

Keywords: Human monkeypoxmonkeypox viruschickenpoxvaricella-zoster virusVZVDemocratic Republic of Congoreal-time PCRhemagglutinin genedispatch

Monkeypox (MPX) virus is an orthopoxvirus that causes human MPX, a smallpoxlike disease reported in the African rainforests. Humans acquire the virus through direct contact with infected animals or patients (1). To determine whether MPX virus could potentially occupy the niche vacated by smallpox virus, the World Health Organization (WHO) conducted an active surveillance program during 1981–1986 in the Democratic Republic of Congo (DRC) and identified 338 MPX cases (67% confirmed by virus culture). Epidemiologic data led to the conclusion that MPX was a sporadic disease with a low potential for person-to-person transmission and that infection could not sustain itself in the human population (1). In the following years (1986–1995), only 13 MPX cases were reported (2). However, during 1995–1996, >500 cases of suspected MPX were reported, although only a small number were laboratory confirmed (3). In contrast to the earlier findings by WHO, the percentage of secondary cases was much higher (78%) and the mortality rate was much lower (1.5%). The question was raised as to whether a number of these cases were actually chickenpox, caused by the varicella-zoster virus (VZV), which is characterized by a high rate of secondary transmission and a low mortality rate (4). Although MPX is the only known severe orthopoxvirus infection today, the current dynamics of MPX infection are poorly understood and little information is available to improve WHO recommendations for the prevention of human MPX. We report laboratory data obtained from patients with suspected MPX infection.

The Study

As part of the DRC Ministry of Health national disease surveillance program, 2,734 cases of suspected human MPX were reported from all 11 DRC provinces during January 2001–December 2004: 380 cases in 2001, 545 in 2002, 783 in 2003, and 1,026 in 2004. However, because civil war severely hampered surveillance activities, only 171 clinical specimens were obtained from 136 patients, who represent 4.9% of all reported cases. Ethical approval for this study was obtained from the Kinshasa School of Public Health, DRC, and the University of North Carolina, Chapel Hill, NC, USA.

All 171 specimens (crusted scabs and vesicle fluids) were inoculated onto MA104-cells by using standard procedures in a Biosafety Level 3 laboratory. Results yielded 56 MPX virus isolates from 48 patients (Table 1); scab and fluid specimens from the same patient were virus-positive for 8 patients. Identity of all isolates as MPX virus was confirmed by sequencing the entire open reading frame of the hemagglutinin (HA) gene.

Orthopoxviruses and varicella-zoster virus, Democratic Republic of Congo, 2001–2004*
Specimen (no. patients)MPX virus isolatedPCR result
OrthopoxvirusVZV
Crusts (50)161821
Vesicle fluid (51)192124
Crusts and vesicle fluid (35)8 and 5†13‡17‡
Total (136)485262

*MPX, monkeypox; VZV, varicella-zoster virus.
†Virus was isolated from both specimens from 8 patients and from either specimen from 5 patients.
‡Both specimens were positive by PCR.

DNA of all specimens was analyzed by using the RealArt Orthopox LightCycler PCR kit (QIAGEN, Hilden, Germany), which amplifies sequences of the fusion protein gene present in all orthopoxviruses, including MPX virus (5). Subsequent melting-curve analysis enables further differentiation of either variola or nonvariola orthopoxviruses; however, specific identification of MPX virus is not possible. We amplified nonvariola orthopoxvirus sequences in 65 specimens from 52 patients (Table 1); all specimens from which MPX virus had been isolated were also positive by PCR. For all 13 patients from whom crusts and vesicle fluids were available, both specimens were PCR-positive.

Of 171 specimens examined by using RealArt VZV LC PCR (QIAGEN), 78 showed specific amplification that indicated that 62 patients had VZV infection (Table 1). Of 136 patients investigated, 1 was coinfected with both, MPX (PCR and virus isolation) and VZV (PCR). Coinfections were reported during the 1996–1997 outbreak (3) and in a patient who died in 2001 (6). Whether these coinfections resulted from simultaneous circulation of both viruses or to a mutual influence in the pathogenesis of either virus is not known.

Data on sex and age were available for 134 patients. The male-to-female ratio was approximately equal: 66 males and 68 females (Table 2). Ages ranged from 2 months to 54 years; average was 15.4 years (median 11.0). The average age of patients with confirmed MPX was 10.0 years (median 7.0 years), whereas the mean age of patients with VZV infection was 20.6 (median 17.0, p<0.001, t = 4.625, df = 109). That most MPX cases (94%) occurred in patients <25 years of age suggests that cross-protective immunity may still exist; a potential for continued immunity has been demonstrated (7). However, this difference could also reflect different exposures in adults >25 years of age.

Age and sex distribution of patients with monkeypox or chickenpox, Democratic Republic of Congo, 2001–2004*
Age, yNo. cases investigated male/female, n = 134MPX-positive male/female, n = 51VZV-positive male/female, n = 61
<412/218/73/6
5–1422/1912/97/8
15–2417/135/7†10/5†
25–349/82/05/6
>356/71/05/6
Total66/6828/2330/31

*MPX, monkeypox; VZV, varicella-zoster virus.
†Coinfection with MPX and VZV in a 17-year-old female patient.

The DRC is divided into 512 administrative health zones that are within 11 provinces (Figure 1). Consistent with earlier reports (1), all MPX patients identified in this study lived mainly in small villages located in or near the tropical rainforest, where populations have ample opportunities for multiple close contacts with animals. No MPX cases have been identified in the area of Kinshasa. In some health zones, MPX virus and VZV circulate simultaneously (data not shown).

Distribution of 52 confirmed cases of human monkeypox (MPX) by health zone in the Democratic Republic of Congo (DRC), 2001–2004. The cumulative number of cases per province is in parentheses. Confirmed cases of MPX originated from a total of 26 different health zones located in 5 provinces of DRC; most (70.5%) were reported from Kasai Oriental and Equateur Provinces. Two groups can be differentiated on the basis of sequence of the hemagglutinin gene: light gray, group 1; dark gray, group 2. Note: The boundaries of the DRC health zones have since been redrawn. Although the health zones Tshopo and Mweka are not shown (located in provinces Orientale and Kasai Occidental, respectively), the general area is highlighted to represent MPX cases in these regions.

The entire sequence (942 nt) of the open reading frame of the HA gene was determined (8) for 48 virus isolates derived from 48 patients after PCR amplification. In addition, the HA gene sequence was successfully determined directly from lesion material of 2 MPX patients from whom no virus isolate was available; for a third patient, only a part of the HA sequence could be sequenced. All HA nucleotide sequences were deposited in GenBank (accession nos. DQ443476 through DQ443525). Sequencing identified 2 distinct groups consisting of 29 and 21 identical sequences, which differed by a single nucleotide. Geographic data analysis demonstrated that this difference was distributed consistently between patients living in east and west DRC (Figure 1). There were no sequence differences in the MPX virus hemagglutinin gene (942 nt) of isolates from the same patient. The 2 sequences determined here were identical to sequences reported previously (9) from MPX patients in the DRC (AF375099 and AF375096, respectively). The sequences represent 2 of 5 branches of the Central African MPX virus clade (Figure 2), which is distinguishable from a second clade comprising isolates from 1) West Africa, 2) outbreaks in primate-holding facilities in Europe and the United States, and 3) the recent 2003 US outbreak.

Phylogenetic inference relationships of the open reading frames encoding the hemagglutinin protein of selected strains of vaccinia, variola, cowpox, and monkeypox viruses and monkeypox virus isolates described in this study. Sequences used were cowpox virus Brighton (AF375089), variola virus Bangladesh (AF375129), vaccinia virus Lister (AY678276), monkeypox virus mpv 1997 (AF375096), mvp-squir (AF375112), mpv Zaire 77-0666 (Z99052), mpv-cncr (AF375102), mpv 74-226 (AF375099), mpv-082 (AF375095), mpv-utc (AF375113), and mpv-3945 (AF375098). ClustalW, version 1.83 (10), was used to generate amino acid multiple sequence alignments (pairwise gap opening = 35 and gap extension = 0.75; multiple alignment gap opening = 15 and gap extension = 0.30; Gonnet series). Each alignment was processed using RevTrans (11). Bayesian posterior probability inference of phylogeny used MrBayes, version 3.084. MrBayes settings for the best-fit model (GTR+I+G) were selected by hierarchies for the likelihood ratio test in MrModeltest 2.0 (12). Bayesian analysis was performed with MrBayes; the maximum likelihood model used 6 substitution types (nst = 6). Rate variation across sites was modeled by using a gamma distribution, with a proportion of sites being invariant (rates = invgamma). The Markov chain Monte Carlo search was run for 1 million generations; trees were sampled every 100 generations (the first 4,000 trees were discarded as burn-in). NL, the Netherlands; DRC, Democratic Republic of Congo.

For our study, we used 2 commercially available kits to amplify orthopoxvirus and VZV sequences. The assays contain an internal control to monitor inhibition and use 4 positive controls to enable quantification of the amount of input genomic viral DNA. On the basis of our results, one can calculate that MPX lesion material usually contains several million viral genomic copies (data not shown). Whereas WHO surveillance confirmed 67% by virus culture (1), we isolated MPX virus in 94% of the MPX patients who had positive PCR results. The rather high rate of virus culture–confirmed cases points to the robust and highly concentrated amounts of virus in lesions; virus remained viable in lesions over an extended period despite suboptimal collection and transportation conditions.

Conclusions

Among 136 patients, 51 (37.5%) had laboratory-confirmed MPX infection, 61 (44.8%) had laboratory-confirmed VZV infection, and 1 (0.7%) had coinfection. MPX virus is considered to be the most important orthopoxvirus infection in humans because it causes disease clinically indistinguishable from smallpox. Recent outbreaks of MPX in the United States (13), the Republic of Congo (14) and Sudan (15) highlight the capacity of this virus to appear where it has never before been reported. Because clinically, MPX is often confused with chickenpox, laboratory diagnosis is critical to determine whether a suspected case is indeed MPX. In our study, we identified the causative agent for a rash-causing illness in 83% of all patients. We have recently strengthened the MPX surveillance program to improve understanding of the epidemiology of human MPX.

Suggested citation for this article: Rimoin AW, Kisalu N, Kebela-Ilunga B, Mukaba T, Wright LL, Formenty P, et al. Endemic human monkeypox, Democratic Republic of Congo, 2001–2004. Emerg Infect Dis [serial on the Internet]. 2007 Jun [date cited]. Available from http://www.cdc.gov/eid/content/13/6/934.htm

Acknowledgments

We respectfully thank the Congolese Ministry of Health and local health workers who were responsible for specimen collection and case investigation. We are very grateful to the Minister of Health of DRC and to Leonard Tapsoba for their constant encouragement and support throughout the study.

Financial support for this study was provided by the National Institutes of Health, National Institute of Child Health and Human Development, Bethesda, Maryland, USA.

Dr Rimoin is an assistant professor in the Department of Epidemiology at the University of California School of Public Health in Los Angeles. Her research interest is the epidemiology of emerging diseases of animal origin, with a particular focus on human monkeypox in the Democratic Republic of Congo.

ReferencesJezek Z, Fenner F Human Monkeypox. In: JL Melnick, editor. Monographs in virology. Volume 17. Basel: Karger; 1988Centers for Disease Control and Prevention Human monkeypox—Kasai Oriental, Democratic Republic of Congo, February 1996–October 1997.MMWR Morb Mortal Wkly Rep 1997;46:1168719408046Hutin YJ, Williams RJ, Malfait P, Pebody R, Loparev VN, Ropp SL, Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997.Emerg Infect Dis 2001;7:434811384521Di Giulio DB, Eckburg PB Human monkeypox: an emerging zoonosis.Lancet Infect Dis 2004;4:1525 10.1016/S1473-3099(03)00856-914720564Olson VA, Laue T, Laker MT, Babkin IV, Drosten C, Shelkunov SN, Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus.J Clin Microbiol 2004;42:19406 10.1128/JCM.42.5.1940-1946.200415131152Meyer H, Perrichot M, Stemmler M, Emmerich P, Schmitz H, Varaine F, Outbreaks of disease suspected of being due to human monkeypox virus infection in the Democratic Republic of Congo in 2001.J Clin Microbiol 2002;40:291921 10.1128/JCM.40.8.2919-2921.200212149352Hammarlund E, Lewis MW, Carter S, Amanna I, Hansen SG, Strelow LI, Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox.Nat Med 2005;11:10051116086024Damaso CR, Esposito JJ, Condit RC, Moussatche N An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine.Virology 2000;277:43949 10.1006/viro.2000.060311080491Chen N, Li G, Liszewski MK, Atkinson JP, Jahrling PB, Feng Z, Virulence differences between monkeypox virus isolates from West Africa and the Congo basin.Virology 2005;340:4663 10.1016/j.virol.2005.05.03016023693Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 1997;25:487682 10.1093/nar/25.24.48769396791Wernersson R, Pedersen AG RevTrans: multiple alignment of coding DNA from aligned amino acid sequences.Nucleic Acids Res 2003;31:35379 10.1093/nar/gkg60912824361Nylander JA, Ronquist F, Huelsenbeck JP, Nieves-Aldrey JL Bayesian phylogenetic analysis of combined data.Syst Biol 2004;53:4767 10.1080/1063515049026469914965900Reed KD, Melski JW, Graham MB, Regnery RL, Sotir MJ, Wegner MV, The detection of monkeypox in humans in the Western Hemisphere.N Engl J Med 2005;350:34250 10.1056/NEJMoa032299Learned LA, Reynolds MG, Wassa DW, Li Y, Olson VA, Karem K, Extended interhuman transmission of monkeypox in a hospital community in the Republic of the Congo, 2003.Am J Trop Med Hyg 2005;73:4283416103616Damon IK, Roth CE, Chowdhary V Discovery of monkeypox in Sudan.N Engl J Med 2006;355:9623 10.1056/NEJMc06079216943415