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1.  Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo 
PLoS Neglected Tropical Diseases  2015;9(6):e0003833.
Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV.
Methodology and Findings
In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds.
This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.
Author Summary
Ebola and Marburg viruses cause haemorrhagic fevers often fatal to humans. Here, we looked for antibodies to Ebola and Marburg viruses (i.e., markers of previous contact with these viruses) in Congolese blood donors with no previous history of haemorrhagic fever. We found serologic evidence for contact with Marburg and Ebola viruses in 0.5% and 2.5% of blood donors, respectively. The circulation of Marburg virus occurs at a very low rate without any identified risk factor. In contrast, prevalence to Ebola virus was peaking at 4% in rural areas and in Pointe Noire city. Importantly, we identified that contacts with bats and birds constituted two independent environmental determinants of exposure. This study confirms that contact with Ebola virus is not infrequent in Congo and can occur in the absence of haemorrhagic fever. It highlights the requirement for further investigating the role of bats and birds in the ecological cycle of Ebola, and for determining whether asymptomatic contact with Ebola virus can provide subsequent protection against severe forms of the Ebola disease.
PMCID: PMC4457487  PMID: 26047124
2.  O’nyong-nyong Virus Infection Imported to Europe from Kenya by a Traveler 
Emerging Infectious Diseases  2014;20(10):1766-1767.
PMCID: PMC4193281  PMID: 25271361
O’nyong-nyong, viruses, Kenya, Germany, mosquito, PCR; Chikungunya
3.  Crimean-Congo Hemorrhagic Fever Virus Clades V and VI (Europe 1 and 2) in Ticks in Kosovo, 2012 
Despite being a small country, Kosovo represents one of the few foci of Crimean-Congo hemorrhagic fever (CCHF) in Europe. The distribution of Kosovar tick vectors and the evolution of CCHF virus in ticks are both as yet unknown. A better description of the extent and the genetic diversity of CCHFV in ticks from endemic settings is essential, in order to be controlled. We investigated the 2012 distribution of Kosovar ticks alongside the prevalence and the phylogeography of tick-derived CCHFV. Hyalomma marginatum dominated in the endemic municipalities with 90.2% versus 24.3% in the non-endemic regions. Of 1,102 tested ticks, 40 (3.6%) were CCHFV-positive, belonging to H. marginatum (29), Rhipicephalus bursa (10), and Ixodes ricinus (1). The virus strains clustered with clade V and VI related sequences. They fell into two lineages: Kosovo I and II. Kosovo I comprised strains recovered exclusively from R. bursa ticks and was closely related to AP92 prototype strain. Kosovo II clustered into Kosovo IIa, including human-derived strains, and IIb including only strains detected in H. marginatum and I. ricinus. Our phylogeographic reconstruction suggests two temporally distinct CCHFV introductions: the most probable location of the most recent common ancestor of Kosovo I lineage was in Greece (63 years ago) and that of lineages IIa-b in Turkey (35 years ago). After each CCHFV introduction into Kosovo, subsequent lineage expansions suggest periods of in situ evolution. The study provides the first insight into the genetic variability and the origin of CCHFV in ticks from Kosovo. Our findings indicate the spreading of CCHFV to non-endemic areas, which underlines the importance of further studies in order to monitor and predict future CCHF outbreaks in Kosovo. The AP92-like strains appear to be more widespread than previously thought and may provide a promising target for experimental studies due to their assumed low pathogenicity.
Author Summary
Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne virus affecting humans. Its distribution coincides with that of Old World Hyalomma tick vectors. The virus circulates in an enzootic cycle, where hard ticks are both vectors and reservoirs, and mammals the amplifying hosts. Accidentally, humans become infected as dead-end hosts. One of the few CCHF-endemic European countries is Kosovo, where an annual average of ten sporadic cases, with a mean mortality rate of 18%, are reported. In this study, the tick distribution, CCHFV prevalence and the phylogeography of the tick-derived strains from 2012 were investigated. The majority of ticks belonged to Hyalomma marginatum, the main vector of CCHFV. Indeed, of 40 CCHFV-positive ticks, 29 were H. marginatum. The phylogenetic analyses revealed that 30 viral strains formed a distinct lineage within clade V (Europe 1) and were closely related to previously known strains from humans and ticks from the Balkans. However, ten isolates from Rhipicephalus bursa ticks, collected in a non-endemic region, turned out to cluster within a very divergent CCHFV clade (Europe 2), so far only known from Greece and Turkey. Since these strains presumably develop only subclinical or mild infections in humans, they might be suitable for experimental vaccine studies.
PMCID: PMC4177860  PMID: 25255381
4.  Genome Analysis of Mayaro Virus Imported to Germany from French Guiana 
Emerging Infectious Diseases  2014;20(7):1255-1257.
PMCID: PMC4073840  PMID: 24960052
Mayaro virus; viruses; genome analysis; travel; Germany; French Guiana
5.  Evaluation of Antiviral Efficacy of Ribavirin, Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever 
Mice lacking the type I interferon receptor (IFNAR−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.
Methodology/Principal Findings
CCHF virus-infected IFNAR−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6–2.8 µg/ml; IC90 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects.
Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.
Author Summary
Crimean-Congo hemorrhagic fever (CCHF) is endemic in Africa, Asia, southeast Europe, and the Middle East. The case fatality rate is 30–50%. Studies on pathophysiology and treatment of CCHF have been hampered by the lack of an appropriate animal model. We have employed CCHF virus-infected transgenic mice, which are defective in the innate immune response, as a disease model. These mice die from the infection and show signs of disease similar to those found in humans. First, we studied the liver pathology in the animals, as hepatic necrosis is a prominent feature of human CCHF. Secondly, we used the model to test the efficacy of antiviral drugs that are in clinical use or in an advanced stage of clinical testing. Besides ribavirin, the standard drug for treatment of CCHF, we tested arbidol, a drug in clinical use against respiratory infections, and T-705, a new drug in clinical development for the treatment of influenza virus infection. While ribavirin and arbidol showed some or no beneficial effect, respectively, T-705 was highly efficacious in the animal model. These data hold promise for clinical efficacy of T-705 in human CCHF.
PMCID: PMC4006714  PMID: 24786461
6.  Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA 
Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.
In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.
Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.
The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.
Author Summary
Infections with four different dengue viruses are threatening 2.5 billion people in tropical countries. Since most antibodies to these four viruses are cross-reacting, a type-specific ELISA would be valuable to study the immune response to the circulating viruses in patients but also in healthy subjects in endemic counties. Therefore a novel DENV immune complex binding (ICB) ELISA was developed to detect serotype-specific antibodies to all four dengue virus serotypes in human serum samples. The tests use labeled recombinant EDIII antigens of the four DENV strains. Numerous samples of patients with RT-PCR confirmed dengue fever were assessed by the new method. In samples of 55 patients with primary dengue fever full agreement between the serotype detected by RT-PCR and the serotype-specific antibody based on the ICB ELISA was obtained. The type-specific antibodies were not observed before the second week of illness. Our data suggest that using the ICB ELISA in healthy adult subjects in an endemic region (Vietnam) both primary and secondary infections can be identified. The method may help to analyze the distribution of the four dengue viruses in the tropics.
PMCID: PMC3873247  PMID: 24386498
7.  Prolonged polyarthralgia in a German traveller with Mayaro virus infection without inflammatory correlates 
BMC Infectious Diseases  2013;13:369.
Mayaro virus is endemic in South America and sporadic outbreaks have been described. It causes a dengue-like febrile illness accompanied by severe and long-lasting polyarthralgias. Outside endemic regions, however, the disease is not well known and can be misdiagnosed as dengue. International travellers are at risk to acquire Mayaro virus and due to increased worldwide travel infectious disease specialists need to be aware of such rare clinical entities.
Case presentation
We report the first Mayaro virus infection imported into Germany. A 20-year-old woman developed fever, myalgia, maculopapular rash, and polyarthralgias following a 10-day trip in the Rurrenabaque region of Bolivia. Severe polyarthralgias persisted for 5 months and were treated with non-steroidal anti-inflammatory drugs. Serological analysis demonstrated Mayaro virus-specific-IgM and -IgG antibodies two months after onset of symptoms. Except for CXCL8/IL-8 other proinflammatory chemokines and cytokines were unremarkable at this time.
Dissemination of knowledge on rare disease might improve patient management. Understanding the inherent features of Mayaro virus infection and how the virus interacts with its host are essential for optimal patient care and therapy.
PMCID: PMC3750572  PMID: 23927600
Mayaro virus; Alphavirus; Persistent arthralgia; Inflammatory cytokines
8.  West Nile Virus Meningoencephalitis Imported into Germany 
Emerging Infectious Diseases  2012;18(10):1698-1700.
PMCID: PMC3471623  PMID: 23017806
West Nile virus; arbovirus; Germany; serology; viruses; vector-borne infections
10.  Epizootic Emergence of Usutu Virus in Wild and Captive Birds in Germany 
PLoS ONE  2012;7(2):e32604.
This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.
PMCID: PMC3289667  PMID: 22389712
11.  Detection of All Species of the Genus Alphavirus by Reverse Transcription-PCR with Diagnostic Sensitivity▿  
Journal of Clinical Microbiology  2010;48(9):3386-3387.
Clinical arbovirus screening requires exclusion of a broad range of viruses with as few assays as possible. We present a reverse transcription-PCR (RT-PCR) for the detection of all species of the genus Alphavirus qualified for exclusion screening (limit of detection [LOD], 5 to 100 RNA copies per reaction across all Alphavirus species; detection of viremia down to ca. 10,000 copies per ml).
PMCID: PMC2937745  PMID: 20504990
12.  Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿  
Journal of Clinical Microbiology  2010;48(6):2009-2013.
The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.
PMCID: PMC2884523  PMID: 20351210
13.  Long-Term Survival of an Urban Fruit Bat Seropositive for Ebola and Lagos Bat Viruses 
PLoS ONE  2010;5(8):e11978.
Ebolaviruses (EBOV) (family Filoviridae) cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts with case fatality rates of up to 90%. Fruit bats may act as reservoirs of the Filoviridae. The migratory fruit bat, Eidolon helvum, is common across sub-Saharan Africa and lives in large colonies, often situated in cities. We screened sera from 262 E. helvum using indirect fluorescent tests for antibodies against EBOV subtype Zaire. We detected a seropositive bat from Accra, Ghana, and confirmed this using western blot analysis. The bat was also seropositive for Lagos bat virus, a Lyssavirus, by virus neutralization test. The bat was fitted with a radio transmitter and was last detected in Accra 13 months after release post-sampling, demonstrating long-term survival. Antibodies to filoviruses have not been previously demonstrated in E. helvum. Radio-telemetry data demonstrates long-term survival of an individual bat following exposure to viruses of families that can be highly pathogenic to other mammal species. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions, further studies should determine if this species forms a reservoir for EBOV from which spillover infections into the human population may occur.
PMCID: PMC2915915  PMID: 20694141
14.  Laboratory Diagnosis of Lassa Fever, Liberia 
Emerging Infectious Diseases  2010;16(6):1041-1043.
PMCID: PMC3086251  PMID: 20507774
Lassa fever; Liberia; laboratory diagnosis; reverse transcription–PCR; viruses; sensitivity; letter
15.  Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands 
Emerging Infectious Diseases  2009;15(8):1171-1175.
Adventure tourism may bring this disease to Western countries.
On July 10, 2008, Marburg hemorrhagic fever was confirmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classification of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact.
PMCID: PMC2815969  PMID: 19751577
Marburg virus diseases; hemorrhagic fever; exposure; contacts; temperature monitoring; filovirus; viruses; perspective
17.  Chikungunya Fever in Travelers Returning to Europe from the Indian Ocean Region, 2006 
Emerging Infectious Diseases  2008;14(3):416-422.
Chikungunya fever should be added to the list of differential diagnoses for ill travelers returning from this region.
Chikungunya fever has spread through several Indian Ocean islands and India, including popular travel destinations. To compare usefulness of diagnostic tests and to understand reasons for the magnitude and severity of an outbreak, we used 3 diagnostic methods to test 720 samples from 680 patients returning to Europe from the Indian Ocean region in 2006. Chikungunya infection was confirmed for 24.4% patients in the first half of the year and for 9.9% in the second half. Reverse transcription–PCR was positive for all samples taken up to day 4 after symptom onset. Immunofluorescence detected immunoglobulin (Ig) M on day 1 and IgG on day 2 for some patients, and in all patients from day 5 onward. Soon after onset of symptoms, patients had IgG and IgM and high viral loads (some >109 copies/mL plasma). These data will help healthcare providers select diagnostic tests for returning travelers.
PMCID: PMC2570846  PMID: 18325256
chikungunya; travelers; dengue; arbovirus diagnostics; real-time RT-PCR; serology; research
18.  Hantavirus Disease Outbreak in Germany: Limitations of Routine Serological Diagnostics and Clustering of Virus Sequences of Human and Rodent Origin▿  
Journal of Clinical Microbiology  2007;45(9):3008-3014.
In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, “Bavaria,” circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.
PMCID: PMC2045283  PMID: 17626170
19.  Lassa Fever, Nigeria, 2003 and 2004 
Emerging Infectious Diseases  2005;11(10):1642-1644.
PMCID: PMC3366737  PMID: 16355508
Lassa fever; Nigeria; arenavirus; letter
20.  Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus 
Journal of Clinical Microbiology  2004;42(5):2043-2047.
First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits—the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)—and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 × 106 and 2.8 × 106 copies/ml (sputum and endotracheal aspirates), 4.3 × 104 and 5.5 × 104 copies/ml (stool), and 5.5 × 102 and 5.2 × 102 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.
PMCID: PMC404639  PMID: 15131168
21.  Outbreaks of Disease Suspected of Being Due to Human Monkeypox Virus Infection in the Democratic Republic of Congo in 2001 
Journal of Clinical Microbiology  2002;40(8):2919-2921.
Seven outbreaks of disease characterized by a pustular rash and suspected to have been caused by human monkeypox virus were investigated. The outbreaks occurred between February and August 2001 in the province of Equateur in the Democratic Republic of Congo. The outbreaks involved a total of 31 persons and caused five deaths. Specimens from 14 patients were available and were analyzed by electron microscopy, virus isolation, and PCR assays specific for monkeypox virus and varicella-zoster virus. We provide evidence that two outbreaks were indeed caused by monkeypox virus (16 cases, with four deaths), that in two outbreaks both monkeypox and varicella-zoster virus were involved (seven cases, with one death), and that two outbreaks were cases of chickenpox caused by infection with varicella-zoster virus (six cases, with no deaths). In one outbreak, no evidence for either monkeypox or chickenpox was found (two cases, with no deaths).
PMCID: PMC120683  PMID: 12149352
22.  Crimean-Congo Hemorrhagic Fever in Kosovo 
Journal of Clinical Microbiology  2002;40(3):1122-1123.
PMCID: PMC120293  PMID: 11880460
23.  Detection of Dengue Virus RNA in Patients after Primary or Secondary Dengue Infection by Using the TaqMan Automated Amplification System 
Journal of Clinical Microbiology  1999;37(8):2543-2547.
In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 × 106 dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).
PMCID: PMC85278  PMID: 10405398

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