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Belgrade Virus, Bulgaria LETTERS Genetic Detection cells/L, hematocrit 51%, blood he- were present. The patient became an- moglobin 161 g/L, platelet count 10 × uric and underwent hemodialysis. On of Dobrava/ 109 cells/L, aspartate aminotransfer- May 9, the patient died. Belgrade Virus, ase (AST) 118 U/L, alanine amino- Serum samples from these 3 pa- Bulgaria transferase (ALT) 89 U/L, urea 26.4 tients were tested for immunoglobulin mmol/L, and creatinine 501 μmol/L. (Ig) G and IgM against Hantaan virus To the Editor: Hantaviruses An echograph showed enlarged kid- (HTNV) and Puumala virus by ELISA (family Bunyaviridae, genus Hanta- neys, liver, spleen, and pancreas, and (Progen, Biotechnik GmbH, Heidel- virus) cause 2 clinical syndromes in abdominal and bilateral pleural effu- berg, Germany). High titers of HTNV humans: hemorrhagic fever with renal sions. Urine analysis disclosed pro- IgM were detected in all 3 patients; in syndrome (HFRS) in the Old World teinuria and microscopic hematuria. 1 patient HTNV IgG was also detect- and hantavirus pulmonary syndrome The patient was admitted with acute ed; antibodies against Puumala virus in the New World (1). Humans are renal failure and multiple organ in- were not detected. Thus, a HTNV-like infected by inhaling the excreta of in- suffi ciency. Despite multiple blood infection was suggested. fected rodents. Persons at increased transfusions and hemodialysis, he Viral RNA was extracted from risk for infection are farmers, loggers/ died 14 days after hospitalization. the earliest available serum sample, forest workers, and soldiers. On April 9, 2010, a 54-year-old and a 1-step SYBR Green real time Bulgaria is a country in southeast- man, a resident of Kirkovo village reverse transcription–PCR (RT-PCR) ern Europe with 8 million inhabitants. (Kardjali Province, southern Bul- (Bio-Rad, Hercules, CA, USA) (2) Two types of hemorrhagic fevers are garia), was admitted to the regional and 2 nested RT-PCRs amplifying par- endemic to Bulgaria: Crimean-Congo hospital in Kardjali City with a 7-day tial small (S) and medium (M) RNA hemorrhagic fever and HFRS. Both history of fever, weakness, and my- segments were applied (3,4). Do- diseases have been subject to manda- algia in the lower extremities and a brava/Belgrade virus (DOBV) RNA tory reporting since 1953. During the 4-day history of abdominal pain and was detected by RT-PCR. Sequenc- past decade, 36 cases of HFRS have diarrhea. At admission, physical ex- ing and phylogenetic analysis of the been reported, mainly in the areas of amination showed skin petechiae, nested RT-PCR products showed that the Balkan and Rila-Pirin-Rodopa subconjunctival and gingival hemor- the causative agent in all 3 cases was mountain ranges in southwestern Bul- rhages, and oliguria. Laboratory fi nd- DOBV (Figure). garia. Diagnosis was based on clinical ings showed the following: leukocyte Sequences were submitted to Gen- symptoms and serologic test results. count of 23 × 109 cells/L, platelet Bank under accession nos. HQ174468– We report 3 HFRS cases, 2 of which count of 50 × 109 cells/L, AST 96 U/L, HQ174473. Bulgarian sequences clus- were fatal. Apart from serologic diag- ALT 167 U/L, urea 58.7 mmol/L, and ter with respective sequences retrieved nosis, genetic detection of hantaviruses creatinine 1,033 μmol/L. Urea and from Apodemus fl avicollis mouse tis- was also achieved, resulting in gaining creatinine levels continued to rise. sues or from HFRS cases from central insight into the genetic relationships Proteinurua and hematuria were pres- and southeastern Europe. Briefl y, the of hantavirus sequences from Bulgaria ent. After 3 sessions of hemodyalisis, closest genetic strains in S and M RNA with respective sequences retrieved in the patient gradually improved, and he segments are strains isolated from A. neighboring countries. was discharged without sequelae. fl avicollis mice in northeastern Greece, On September 2, 2009, high fe- On May 7, 2010, a 28-year-old near the border with Bulgaria (5). The ver, chills, headache, and myalgia man, a resident of Smilyan village, genetic difference at nucleotide level developed in a 21-year-old man who (Smolyan Province, southern Bul- among the Bulgarian strains is 1.2%– lived in Simitli town (Blagoevgrad garia) was admitted to the Infectious 2.1% and 2.2%–7.4% in the S and M Province, southwestern Bulgaria). Diseases Clinic in Smolyan Regional segments, respectively. Five days after symptom onset, he Hospital with a 4-day history of fe- HFRS is endemic to the Bal- was admitted to the regional hospital ver, vomiting, and diarrhea. Physical kan Peninsula. Severe HFRS cases of Blagoevgrad. His condition rap- examination on admission showed caused by DOBV have been reported idly deteriorated. Clinical signs were skin petechiae and gingival hemor- in Greece (4–6), Slovenia (7,8), Ser- pharyngeal hyperemia, oliguria, and rhages. Laboratory fi ndings showed bia and Montenegro (9), the Czech febrile toxic syndrome. The patient the following: leukocyte count of 6 × Republic (3), and Hungary (10). Our became hypotensive, reporting ab- 109 cells/L, platelet count of 50 × 109 results confi rm that DOBV also cir- dominal pain in the liver and spleen. cells/L, urea 10.5 mmol/L, creatinine culates in Bulgaria and causes severe Laboratory fi ndings showed the fol- 230 mmol/L, AST 1697 U/L, and ALT HFRS cases; thus, clinicians have to lowing: leukocyte count 11.8 × 109 1,119 U/L. Proteinuria and hematuria include HFRS in differential diagnosis 308 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 2, February 2011 LETTERS 9. Papa A, Bojovic B, Antoniadis A. Hanta- viruses in Serbia and Montenegro. Emerg Infect Dis. 2006;12:1015–8. 10. Jakab F, Sebok J, Ferenczi E, Horvath G, Szucs G. First detection of Dobrava hantavirus from a patient with severe hae- morrhagic fever with renal syndrome by SYBR Green-based real time RT-PCR. Scand J Infect Dis. 2007;39:902–6. DOI: 10.1080/00365540701387072 Address for correspondence: Anna Papa, Department of Microbiology, Medical School Aristotle University of Thessaloniki, 54124, Thessaloniki 999149, Greece; email: annap@ med.auth.gr Figure. Phylogenetic trees based on a 560-bp fragment of the small RNA segment (A) and a 224-bp fragment of the medium RNA segment (B) of hantaviruses. Hantaan virus (HTNV) was used as the outgroup. The numbers at the nodes indicate percentage bootstrap replicates of 100; values <60% are not shown. Horizontal distances are proportional to the nucleotide differences. Sequences in the tree are indicated as GenBank accession number, strain name, country. Strains from this study are shown in boldface. Scale bars indicate 10% nucleotide sequence divergence. A226V Strains of Chikungunya Virus, of febrile cases accompanied by acute J Virol Methods. 2005;124:21–6. DOI: Réunion Island, nephropathy. Further studies on pa- 10.1016/j.jviromet.2004.10.004 3. Papa A, Zelena H, Barnetova D, Petrous- 2010 tients and small mammals in Bulgaria ova L. Genetic detection of Dobrava/Bel- will elucidate the hantavirus epidemi- grade virus in a Czech patient with haem- To the Editor: Chikungunya vi- ology in this Balkan region. orrhagic fever with renal syndrome. Clin rus (CHIKV) fi rst emerged in Indian Microbiol Infect. 2010;16:1187–90. DOI: 10.1111/j.1469-0691.2009.03075.x Ocean islands off the eastern coast of Acknowledgments 4. Papa A, Johnson AM, Stockton PC, Africa in 2005 and was responsible for We thank N. Kalvatchev and E. Pa- Bowen MD, Spiropoulou CF, Alexiou- large-scale epidemics on the islands of padimitriou for excellent technical assis- Daniel S, et al. Retrospective serologi- Réunion, Comoros, Mayotte, Mau- cal and genetic study of the distribution tance. of hantaviruses in Greece. J Med Virol. ritius, Madagascar, and Seychelles 1998;55:321–7. DOI: 10.1002/(SICI)1096- (1–4). On Réunion Island, a French Anna Papa and Iva Christova 9071(199808)55:4<321::AID- overseas territory of 810,000 inhabit- JMV11>3.0.CO;2-H ants, herd immunity reached 38% in Author affi liations: Medical School, Aristotle 5. Papa A, Nemirov K, Henttonen H, Ni- University of Thessaloniki, Thessaloniki, emimaa J, Antoniadis A, Vaheri A, et al. October 2006 (5). Molecular epide- Greece (A. Papa); and National Centre of Isolation of Dobrava virus from Apode- miology of the strain responsible for mus fl avicollis in Greece. J Clin Micro- Infectious and Parasitic Diseases, Sofi a, these outbreaks indicated that it had biol. 2001;39:2291–3. DOI: 10.1128/ originated in Kenya (6). The epidemic Bulgaria (I. Christova) JCM.39.6.2291-2293.2001 6. Papa A, Antoniadis A. Hantavirus infec- on Réunion Island was associated with DOI: 10.3201/eid1702.101275 tions in Greece–an update. Eur J Epide- a mutation in the envelope protein miol. 2001;17:189–94. DOI: 10.1023/ gene (E1-A226V) that improves rep- A:1017987104363 lication and transmission effi ciency in 7. Avsic-Zupanc T, Petrovec M, Furlan P, References Kaps R, Elgh F, Lundkvist A. Hemor- Aedes albopictus mosquitoes (7). rhagic fever with renal syndrome in the Since 2006, the Regional Offi ce of 1. Jonsson CB, Figueiredo LT, Vapalahti O. A Dolenjska region of Slovenia—a 10-year global perspective on hantavirus ecology, the French Institute for Public Health survey. Clin Infect Dis. 1999;28:860–5. epidemiology, and disease. Clin Micro- Surveillance in the Indian Ocean has DOI: 10.1086/515185 biol Rev. 2010;23:412–41. DOI: 10.1128/ 8. Saksida A, Duh D, Korva M, Avsic-Zupanc conducted epidemiologic and biologi- CMR.00062-09 T. Dobrava virus RNA load in patients cal surveillance for CHIKV infection. 2. Aitichou M, Saleh SS, McElroy AK, who have hemorrhagic fever with renal Schmaljohn C, Ibrahim MS.
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