Journal of Clinical Virology 61 (2014) 439–441
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Case report
Human Dobrava-Belgrade hantavirus infection, Kosovo Petra Emmerich a,1 , Nicole Müller b,1 , Patrick Heinemann a,c,1 , Enno Rother d , Xhevat Jakupi e , Stephan Günther a , Daniel Cadar a , Jonas Schmidt-Chanasit a,f,∗ a Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Hamburg, Germany b Department of Internal Medicine, German Armed Forces Medical Centre, Hamburg, Germany c Institute of Medical Virology, Charite Medical School, Berlin, Germany d Department of Internal Medicine, German Armed Forces Medical Centre, Ulm, Germany e National Institute of Public Health of Kosovo, Pristina, Kosovo, Serbia f German Centre for Infection Research (DZIF), partner site Hamburg-Luebeck-Borstel, Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 7 May 2014 Received in revised form 20 September 2014 Accepted 22 September 2014 Keywords: Dobrava-Belgrade virus Hantavirus Genome Kosovo HFRS
a b s t r a c t Here we describe an acute Dobrava-Belgrade virus (DOBV) infection that presented as severe hemorrhagic fever with renal syndrome (HFRS) in an active-duty U.S. soldier. The infection was acquired in northern Kosovo in spring 2013. Amplification of DOBV genome segments directly from the patient’s serum sample was successfully performed. Phylogenetic analysis demonstrated that the strain belong to DOBV genotype Dobrava and is closely related to strains circulating in Southeast Europe and Slovakia. Thus, our case confirms that DOBV genotype Dobrava is able to cause a severe form of HFRS, especially when compared to the other less pathogenic DOBV genotypes. © 2014 Elsevier B.V. All rights reserved.
1. Why this case is important Dobrava-Belgrade virus (DOBV), a rodent-borne hantavirus of the Bunyaviridae family, may cause hemorrhagic fever with renal syndrome (HFRS) and was first isolated more than 20 years ago from a yellow-necked mouse (Apodemus flavicollis) in Slovenia [1,2]. Simultaneously, isolation of the same virus from a HFRS patient in Belgrade was reported [3]. DOBV is an enveloped virus with tripartite RNA genome (S, M and L segment) with negative polarity [1]. It is transmitted to humans by inhalation of aerosols that are contaminated with urine, feces, and saliva of infected reservoir rodents [1]. A subdivision of the DOBV into four distinct genotypes (Dobrava, Kurkino, Saaremaa, and Sochi) that are hosted by different Apodemus species and cause HFRS of varying degrees of severity was proposed recently [4]. In 2008, serological investigations for hantavirus-specific antibodies in 203 persons
∗ Corresponding author at: Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany. Tel.: +49 40 428 18 959; fax: +49 40 428 18 941. E-mail address: [email protected] (J. Schmidt-Chanasit). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.jcv.2014.09.013 1386-6532/© 2014 Elsevier B.V. All rights reserved.
from Kosovo demonstrated a seroprevalence of 7.9% [5]. Here we describe an acute DOBV genotype Dobrava infection that presented as severe HFRS. Partial amplification of the three DOBV genome segments directly from the patient’s serum sample was successfully performed.
2. Case report A 37-year old active-duty U.S. soldier was transferred on May 13, 2013 to the German Field Hospital in Prizren from Camp Bondsteel near Ferizaj/Uroˇsevac, Kosovo, because of deterioration and dehydration. The symptoms started four days prior in Camp Novo Selo (located in northern part of Kosovo) with nausea and vomiting during the night followed by fever, headache and watery foul-smelling green diarrhea. The patient operated in northern Kosovo and denied any tick bites or consumption of food outside the camp. Outside the Balkan, the patient has not traveled within the last months. The patient’s past medical history was unremarkable. There were no other soldiers in his unit with similar symptoms. The clinical examination revealed tachypnea (31/min) and mild diffuse abdominal tenderness. No petechial stigmata, nor rash or edema were observed. Laboratory investigations revealed thrombocytopenia (13,000/l), a prolonged partial thromboplastin
440
P. Emmerich et al. / Journal of Clinical Virology 61 (2014) 439–441
Fig. 1. Phylogenetic trees based on Bayesian analysis of the nucleocapsid protein (S segment), glycoprotein (M segment) and RNA-dependent RNA polymerase protein (L segment) coding sequences of Dobrava-Belgrade virus (DOBV). The phylogenetic analyses were performed by using MrBayes 3.1.2 [10] with Bayesian Markov chain Monte Carlo (MCMC) tree-sampling methods based on 2 runs consisting of 4 chains of 1,000,000 with a burn-in of 25% and a parallel maximum-likelihood bootstrap analysis with 1000 pseudo-replicates using PhyML. The Akaike’s Information Criterion was chosen as the model selection framework and General-Time-Reversible model of sequence evolution with gamma-distributed rate variation among sites and a proportion of invariable sites (GTR + + I) for M and S segments and gamma-distributed rate variation among sites (GTR + ) for L segment as the best model. The trees are based on partial S (∼1 kb), M (∼1 kb) and L (∼1.9 kb) segments of DOBV strains. Numbers at the nodes indicate posterior probability values (clade credibilities ≥90%) and parallel Maximum Likelihood bootstrap replicates (≥70%). The DOBV strain (KOS-1) from this study is bolded. The tree was rooted using the following hantaviruses: Haantan virus (HNTV), Sangassou virus (SANGV), Seoul virus (SEOV) and Thailand virus (THAIV). Human and different rodent-derived DOBV viruses are indicated in the threes. Scale bar indicates mean number of nucleotide substitutions per site. GenBank accession numbers and country of origin are shown.
P. Emmerich et al. / Journal of Clinical Virology 61 (2014) 439–441
time (53 s), increased lactate dehydrogenase (458 U/l), proteinuria and hematuria. Due to the headache and fever, cerebrospinal fluid (CSF) puncture was performed to rule out meningitis or encephalitis. CSF analysis (pathogen detection by PCR) revealed no evidence for bacterial or viral meningitis or encephalitis, respectively. Antigen tests for different gastrointestinal viruses were negative and analyses of blood and stool cultures revealed no pathological findings. The abdominal ultrasound showed moderate splenomegaly (16 cm × 5 cm). A CT-scan showed diffuse intra- and extra-peritoneal fluid with a diffuse inflammation of the intestines and a moderate splenomegaly. His respiratory status prompted an evaluation of his acid-base status which revealed a combined disorder. There was a metabolic acidosis likely due to bicarbonate loss from diarrhea and a primary respiratory alkalosis, presumably from sepsis-induced hyperventilation. Thus, the patient was transferred to intensive care unit and got crystalloid fluid, symptomatic treatment of the fever and broad spectrum antibiotics (Tazobactam, Piperacillin and Ciprofloxacin). The patient showed stable vital signs but despite the therapeutic measures the overall status of the patient further deteriorated. The echocardiography showed worsening global left ventricular function consistent with septic cardiomyopathy and thrombocyte count decreased to 9000/l without any signs of bleeding. The most likely diagnosis of sepsis due to a fulminant gastroenteritis was ruled out by laboratory testings. Second, an atypical pneumonia was also unlikely without infiltrates on the CT scan after three days of illness. Because of the proteinuria and hematuria, hantavirus infection was suspected and serological tests (recomLine Bunyavirus IgG/IgM, Mikrogen, Germany) and RT-PCR [6] were performed. Serological analysis revealed a positive result for DOBV IgM and a negative result for DOBV IgG. Results for Hantaan virus, Seoul virus and Puumala virus IgM and IgG were negative. RT-PCR was positive and sequencing of the amplicon revealed a DOBV-specific sequence (L segment) demonstrating an acute DOBV infection. The patient was then transferred to Germany (Landstuhl) for further treatment and presented there with a progressive non-oliguric renal failure with a peak creatinine of 4.8 mg/dl plus respiratory failure requiring intermittent bipap-ventilation. Platelets were given to prevent bleeding and thereafter remained stable. Nine days after disease onset, the patient entered the polyuric phase of HFRS and started to improve. The patient has meanwhile returned to his home country recovering from the DOBV infection with a characterization of restitutio ad integrum. Attempts to isolate DOBV in cell culture (Vero E6 cells) failed. Therefore, the serum sample was subjected for amplification of DOBV S, M and L genome segments. Phylogenetic analysis of partial S, M, and L segments demonstrated that the strain KOS1 belong to DOBV genotype Dobrava (according to Ref. [4]) and is closely related to strains circulating in Southeast Europe and Slovakia (Fig. 1). 3. Other similar cases in the literature and discussion Our case demonstrates that DOBV genotype Dobrava is able to cause a severe form of HFRS, especially when compared to the other less pathogenic DOBV genotypes. Although gastrointestinal symptoms are common, extensive diarrhea like in our patient has not
441
been described in the literature [4,7,8]. Relevant thrombocytopenia occurs in the majority of HFRS patients and thrombocyte levels below 50,000/l develops in more than half of the cases, but the extent of decrease in platelet count observed in our case is unusual [8,9]. Further studies in Kosovo should focus on the identification of the respective reservoir hosts for DOBV. Moreover, extensive epidemiological studies should identify areas in Kosovo with a higher incidence for DOBV infections. Thus, soldiers and the resident population could be better protected from a potentially lethal disease. Funding None. Competing interest None declared. Ethical approval Not required. Conflict of interest The authors do not have commercial or other associations that might pose a conflict of interest (e.g., pharmaceutical stock ownership or consultancy). Acknowledgement This case report is dedicated to the late Ursula Herrmann (1927–2014), for making this study possible. References [1] Krüger DH, Ulrich RG, Hofmann J. Hantaviruses as zoonotic pathogens in Germany. Dtsch Arztebl Int 2013;110(July):461–7. [2] Avsic-Zupanc T, Xiao SY, Stojanovic R, Gligic A, van der Groen G, LeDuc JW. Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia. J Med Virol 1992;38:132–7. [3] Gligic A, Dimkovic N, Xiao SY, Buckle GJ, Jovanovic D, Velimirovic D, et al. Belgrade virus: a new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia. J Infect Dis 1992;166:113–20. [4] Klempa B, Avsic-Zupanc T, Clement J, Dzagurova TK, Henttonen H, Heyman P, et al. Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics. Arch Virol 2013;158:521–9. [5] Muc¸aj S, Kabashi S, Ahmeti S, Dedushaj I, Ramadani N, Avsic-Zupanc T. Collective immunity of the population from endemic zones of hemorrhagic fever with renal syndrome in Kosovo. Med Arh 2009;63(3):160–2. [6] Klempa B, Fichet-Calvet E, Lecompte E, Auste B, Aniskin V, Meisel H, et al. Hantavirus in African wood mouse, Guinea. Emerg Infect Dis 2006;12:838–40. [7] Schmidt-Chanasit J, Meisel H, Hofmann J, Rang A, Lambrecht E, Ulrich RG, et al. Clinical course and laboratory parameters of the first Dobrava-Belgrade hantavirus infection imported to Germany. J Clin Virol 2008;42:91–3. [8] Kuzman I. Clinical picture of hemorrhagic fever with renal syndrome in Croatia. Acta Med Croatia 2003;57:393–7. [9] Markotic´ A, Nichol ST, Kuzman I, Sanchez AJ, Ksiazek TG, Gagro A, et al. Characteristics of Puumala and Dobrava infections in Croatia. J Med Virol 2002;66:542–51. [10] Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003;19:1572–4.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Case report
Human Dobrava-Belgrade hantavirus infection, Kosovo Petra Emmerich a,1 , Nicole Müller b,1 , Patrick Heinemann a,c,1 , Enno Rother d , Xhevat Jakupi e , Stephan Günther a , Daniel Cadar a , Jonas Schmidt-Chanasit a,f,∗ a Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Hamburg, Germany b Department of Internal Medicine, German Armed Forces Medical Centre, Hamburg, Germany c Institute of Medical Virology, Charite Medical School, Berlin, Germany d Department of Internal Medicine, German Armed Forces Medical Centre, Ulm, Germany e National Institute of Public Health of Kosovo, Pristina, Kosovo, Serbia f German Centre for Infection Research (DZIF), partner site Hamburg-Luebeck-Borstel, Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 7 May 2014 Received in revised form 20 September 2014 Accepted 22 September 2014 Keywords: Dobrava-Belgrade virus Hantavirus Genome Kosovo HFRS
a b s t r a c t Here we describe an acute Dobrava-Belgrade virus (DOBV) infection that presented as severe hemorrhagic fever with renal syndrome (HFRS) in an active-duty U.S. soldier. The infection was acquired in northern Kosovo in spring 2013. Amplification of DOBV genome segments directly from the patient’s serum sample was successfully performed. Phylogenetic analysis demonstrated that the strain belong to DOBV genotype Dobrava and is closely related to strains circulating in Southeast Europe and Slovakia. Thus, our case confirms that DOBV genotype Dobrava is able to cause a severe form of HFRS, especially when compared to the other less pathogenic DOBV genotypes. © 2014 Elsevier B.V. All rights reserved.
1. Why this case is important Dobrava-Belgrade virus (DOBV), a rodent-borne hantavirus of the Bunyaviridae family, may cause hemorrhagic fever with renal syndrome (HFRS) and was first isolated more than 20 years ago from a yellow-necked mouse (Apodemus flavicollis) in Slovenia [1,2]. Simultaneously, isolation of the same virus from a HFRS patient in Belgrade was reported [3]. DOBV is an enveloped virus with tripartite RNA genome (S, M and L segment) with negative polarity [1]. It is transmitted to humans by inhalation of aerosols that are contaminated with urine, feces, and saliva of infected reservoir rodents [1]. A subdivision of the DOBV into four distinct genotypes (Dobrava, Kurkino, Saaremaa, and Sochi) that are hosted by different Apodemus species and cause HFRS of varying degrees of severity was proposed recently [4]. In 2008, serological investigations for hantavirus-specific antibodies in 203 persons
∗ Corresponding author at: Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany. Tel.: +49 40 428 18 959; fax: +49 40 428 18 941. E-mail address: [email protected] (J. Schmidt-Chanasit). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.jcv.2014.09.013 1386-6532/© 2014 Elsevier B.V. All rights reserved.
from Kosovo demonstrated a seroprevalence of 7.9% [5]. Here we describe an acute DOBV genotype Dobrava infection that presented as severe HFRS. Partial amplification of the three DOBV genome segments directly from the patient’s serum sample was successfully performed.
2. Case report A 37-year old active-duty U.S. soldier was transferred on May 13, 2013 to the German Field Hospital in Prizren from Camp Bondsteel near Ferizaj/Uroˇsevac, Kosovo, because of deterioration and dehydration. The symptoms started four days prior in Camp Novo Selo (located in northern part of Kosovo) with nausea and vomiting during the night followed by fever, headache and watery foul-smelling green diarrhea. The patient operated in northern Kosovo and denied any tick bites or consumption of food outside the camp. Outside the Balkan, the patient has not traveled within the last months. The patient’s past medical history was unremarkable. There were no other soldiers in his unit with similar symptoms. The clinical examination revealed tachypnea (31/min) and mild diffuse abdominal tenderness. No petechial stigmata, nor rash or edema were observed. Laboratory investigations revealed thrombocytopenia (13,000/l), a prolonged partial thromboplastin
440
P. Emmerich et al. / Journal of Clinical Virology 61 (2014) 439–441
Fig. 1. Phylogenetic trees based on Bayesian analysis of the nucleocapsid protein (S segment), glycoprotein (M segment) and RNA-dependent RNA polymerase protein (L segment) coding sequences of Dobrava-Belgrade virus (DOBV). The phylogenetic analyses were performed by using MrBayes 3.1.2 [10] with Bayesian Markov chain Monte Carlo (MCMC) tree-sampling methods based on 2 runs consisting of 4 chains of 1,000,000 with a burn-in of 25% and a parallel maximum-likelihood bootstrap analysis with 1000 pseudo-replicates using PhyML. The Akaike’s Information Criterion was chosen as the model selection framework and General-Time-Reversible model of sequence evolution with gamma-distributed rate variation among sites and a proportion of invariable sites (GTR + + I) for M and S segments and gamma-distributed rate variation among sites (GTR + ) for L segment as the best model. The trees are based on partial S (∼1 kb), M (∼1 kb) and L (∼1.9 kb) segments of DOBV strains. Numbers at the nodes indicate posterior probability values (clade credibilities ≥90%) and parallel Maximum Likelihood bootstrap replicates (≥70%). The DOBV strain (KOS-1) from this study is bolded. The tree was rooted using the following hantaviruses: Haantan virus (HNTV), Sangassou virus (SANGV), Seoul virus (SEOV) and Thailand virus (THAIV). Human and different rodent-derived DOBV viruses are indicated in the threes. Scale bar indicates mean number of nucleotide substitutions per site. GenBank accession numbers and country of origin are shown.
P. Emmerich et al. / Journal of Clinical Virology 61 (2014) 439–441
time (53 s), increased lactate dehydrogenase (458 U/l), proteinuria and hematuria. Due to the headache and fever, cerebrospinal fluid (CSF) puncture was performed to rule out meningitis or encephalitis. CSF analysis (pathogen detection by PCR) revealed no evidence for bacterial or viral meningitis or encephalitis, respectively. Antigen tests for different gastrointestinal viruses were negative and analyses of blood and stool cultures revealed no pathological findings. The abdominal ultrasound showed moderate splenomegaly (16 cm × 5 cm). A CT-scan showed diffuse intra- and extra-peritoneal fluid with a diffuse inflammation of the intestines and a moderate splenomegaly. His respiratory status prompted an evaluation of his acid-base status which revealed a combined disorder. There was a metabolic acidosis likely due to bicarbonate loss from diarrhea and a primary respiratory alkalosis, presumably from sepsis-induced hyperventilation. Thus, the patient was transferred to intensive care unit and got crystalloid fluid, symptomatic treatment of the fever and broad spectrum antibiotics (Tazobactam, Piperacillin and Ciprofloxacin). The patient showed stable vital signs but despite the therapeutic measures the overall status of the patient further deteriorated. The echocardiography showed worsening global left ventricular function consistent with septic cardiomyopathy and thrombocyte count decreased to 9000/l without any signs of bleeding. The most likely diagnosis of sepsis due to a fulminant gastroenteritis was ruled out by laboratory testings. Second, an atypical pneumonia was also unlikely without infiltrates on the CT scan after three days of illness. Because of the proteinuria and hematuria, hantavirus infection was suspected and serological tests (recomLine Bunyavirus IgG/IgM, Mikrogen, Germany) and RT-PCR [6] were performed. Serological analysis revealed a positive result for DOBV IgM and a negative result for DOBV IgG. Results for Hantaan virus, Seoul virus and Puumala virus IgM and IgG were negative. RT-PCR was positive and sequencing of the amplicon revealed a DOBV-specific sequence (L segment) demonstrating an acute DOBV infection. The patient was then transferred to Germany (Landstuhl) for further treatment and presented there with a progressive non-oliguric renal failure with a peak creatinine of 4.8 mg/dl plus respiratory failure requiring intermittent bipap-ventilation. Platelets were given to prevent bleeding and thereafter remained stable. Nine days after disease onset, the patient entered the polyuric phase of HFRS and started to improve. The patient has meanwhile returned to his home country recovering from the DOBV infection with a characterization of restitutio ad integrum. Attempts to isolate DOBV in cell culture (Vero E6 cells) failed. Therefore, the serum sample was subjected for amplification of DOBV S, M and L genome segments. Phylogenetic analysis of partial S, M, and L segments demonstrated that the strain KOS1 belong to DOBV genotype Dobrava (according to Ref. [4]) and is closely related to strains circulating in Southeast Europe and Slovakia (Fig. 1). 3. Other similar cases in the literature and discussion Our case demonstrates that DOBV genotype Dobrava is able to cause a severe form of HFRS, especially when compared to the other less pathogenic DOBV genotypes. Although gastrointestinal symptoms are common, extensive diarrhea like in our patient has not
441
been described in the literature [4,7,8]. Relevant thrombocytopenia occurs in the majority of HFRS patients and thrombocyte levels below 50,000/l develops in more than half of the cases, but the extent of decrease in platelet count observed in our case is unusual [8,9]. Further studies in Kosovo should focus on the identification of the respective reservoir hosts for DOBV. Moreover, extensive epidemiological studies should identify areas in Kosovo with a higher incidence for DOBV infections. Thus, soldiers and the resident population could be better protected from a potentially lethal disease. Funding None. Competing interest None declared. Ethical approval Not required. Conflict of interest The authors do not have commercial or other associations that might pose a conflict of interest (e.g., pharmaceutical stock ownership or consultancy). Acknowledgement This case report is dedicated to the late Ursula Herrmann (1927–2014), for making this study possible. References [1] Krüger DH, Ulrich RG, Hofmann J. Hantaviruses as zoonotic pathogens in Germany. Dtsch Arztebl Int 2013;110(July):461–7. [2] Avsic-Zupanc T, Xiao SY, Stojanovic R, Gligic A, van der Groen G, LeDuc JW. Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia. J Med Virol 1992;38:132–7. [3] Gligic A, Dimkovic N, Xiao SY, Buckle GJ, Jovanovic D, Velimirovic D, et al. Belgrade virus: a new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia. J Infect Dis 1992;166:113–20. [4] Klempa B, Avsic-Zupanc T, Clement J, Dzagurova TK, Henttonen H, Heyman P, et al. Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics. Arch Virol 2013;158:521–9. [5] Muc¸aj S, Kabashi S, Ahmeti S, Dedushaj I, Ramadani N, Avsic-Zupanc T. Collective immunity of the population from endemic zones of hemorrhagic fever with renal syndrome in Kosovo. Med Arh 2009;63(3):160–2. [6] Klempa B, Fichet-Calvet E, Lecompte E, Auste B, Aniskin V, Meisel H, et al. Hantavirus in African wood mouse, Guinea. Emerg Infect Dis 2006;12:838–40. [7] Schmidt-Chanasit J, Meisel H, Hofmann J, Rang A, Lambrecht E, Ulrich RG, et al. Clinical course and laboratory parameters of the first Dobrava-Belgrade hantavirus infection imported to Germany. J Clin Virol 2008;42:91–3. [8] Kuzman I. Clinical picture of hemorrhagic fever with renal syndrome in Croatia. Acta Med Croatia 2003;57:393–7. [9] Markotic´ A, Nichol ST, Kuzman I, Sanchez AJ, Ksiazek TG, Gagro A, et al. Characteristics of Puumala and Dobrava infections in Croatia. J Med Virol 2002;66:542–51. [10] Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003;19:1572–4.
