Antonis Antoniadis

Crimean-Congo Hemorrhagic Fever in Albania, 2001

Abstract.During the spring and summer of 2001, an outbreak of eight cases of Crimean-Congo hemorrhagic fever (CCHF) occurred in Albania. The epidemiological investigation, the clinical presentation of the cases, and the course of the disease are described. Seven of the cases were laboratory confirmed. A nosocomial infection and a cluster of cases within a family were observed. Genetic analysis of the CCHF virus strain that caused the outbreak showed that it was clustered together with other European CCHF virus strains except the Greek one (strain AP92). The Greek strain, which forms an independent clade, differed from the causative strain by 25.3% at the nucleotide level.

Crimean-Congo Hemorrhagic Fever in Bulgaria

We report the epidemiologic characteristics of Crimean-Congo hemorrhagic fever in Bulgaria, as well as the first genetic characterization of the virus strains circulating in the country in 2002 to 2003 that caused disease in humans.

Retrospective serological and genetic study of the distribution of hantaviruses in Greece

A retrospective serological and genetic study of hantaviruses responsible for hemorrhagic fever with renal syndrome (HFRS) in Greece during the last 17 years is presented. Fifty‐one serum samples taken from 30 HFRS cases previously diagnosed by immunofluorescence assay were tested by ELISA for IgG (Hantaan, Dobrava, and Puumala) and IgM antibodies (Hantaan and Puumala). Results were compatible with the majority of infections being related to hantaviruses carried by rodents of the subfamily Murinae. RNA was extracted from 26 selected samples and reverse transcriptase‐polymerase chain reaction (RT‐PCR) was performed using primers specifically designed for the detection of hantaviruses associated with murine (MS‐N‐specific, MM‐G1‐specific primers) or arvicoline rodents (PPT‐N‐specific primers). In addition, primers previously designed for the detection of the G2 coding region of the Murinae‐associated hantaviruses were also used. Sequencing of the PCR products was then performed, followed by phylogenetic analysis of nucleotide sequence differences. Eleven out of the 26 serum samples tested were found to be positive by PCR with the MS‐N primers, whereas four were positive with the MM‐G1 primers, and only two with the G2 primers. None of the samples was found positive with the PPT primers. The sequence analysis showed that the virus that was responsible for these 11 HFRS cases was the Dobrava virus, which is endemic throughout the Balkans. J. Med. Virol. 55:321–327, 1998.

Genetic Characterization of the M RNA Segment of Crimean Congo Hemorrhagic Fever Virus Strains, China

We report the genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus (CCHFV). Two CCHFV strains isolated in Xinjiang Province, a region endemic for CCHF in northwestern China, were studied. These strains, designated BA66019 and BA8402, were isolated in 1965 and 1984 from a CCHF patient and Hyalomma ticks, respectively. Viral RNA was extracted from suckling mouse brains infected with these two strains, amplified, and sequenced. The full-length M RNA, consisting of 5.3 kb, was determined for both strains. The coding nucleotide sequences of the two strains differed from each other by 17.5% and from the reference CCHFV strain IbAr10200 by a mean of 22%, suggesting that the genus Nairovirus comprises a group of genetically highly diverse strains.

Emergence of Crimean–Congo haemorrhagic fever in Greece

In the summer of 2008, the first case of Crimean-Congo haemorrhagic fever (CCHF) was observed in Greece. The laboratory diagnosis was established using nested RT-PCR and quantitative real-time RT-PCR. A high viral load and increased levels of cytokines were detected on the third day of illness and the patient died 7 days after the onset of symptoms. Nucleotide sequence analysis revealed that the Greek CCHF virus strain had high sequence identity with other Balkan CCHF virus strains.

Aseptic meningitis and encephalitis because of herpesviruses and enteroviruses in an immunocompetent adult population.

Background and aim:  Human herpesviruses (HHVs) and enteroviruses (EVs) are the major causative agents of CNS viral infections. The aim of the study was to identify the etiology and determine the frequency of aseptic meningitis and encephalitis due to HHVs and EVs in an immunocompetent adult population.

Viral load and Crimean-Congo hemorrhagic fever.

To the Editor: Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease transmitted to humans by tick bite or contact with blood, excreta, or tissues of infected patients or livestock. The disease is endemic in many African, Asian, and European countries. Sporadic cases or outbreaks have been observed in the Balkan Peninsula (1–5). Prompt diagnosis of the disease is essential for preventing human-to-human transmission. Reverse transcription–PCR (RT-PCR) is the detection method of choice in first days of illness and in severe cases with no antibody production. In recent years, real-time RT-PCR approaches have been described for detection and quantification of CCHF virus (6–8). However, no information is available on viral RNA concentration in patients. We describe a real-time RT-PCR for detection and quantification of CCHF virus, present the results of its use with clinical samples, and report the relationship between viral load and severity and outcome of CCHF. We tested 29 serum samples from Albanian patients with suspected CCHF or their contacts who were living in a CCHF-endemic area of Albania. Serum samples were collected during 2003–2006 and categorized into 3 groups. Group A contained samples from 11 patients with CCHF confirmed by a conventional RT-nested PCR (9). Group B contained samples from 5 patients who had negative RT-nested PCR results and positive serologic results. Group C contained samples from 15 persons who were from the same region as the CCHF patients but who did not have any clinical symptoms of CCHF and had negative PCR or serologic results. One set of primers and 1 probe were designed to amplify an 84-bp genome region of the S RNA segment of CCHF virus on the basis of European sequences (Balkan and Russian strains available in GenBank): primers CCEuS 5′-TGACAGCATTTCTTTAACAGACATCA-3′ and CCEuAs 5′-AAACACGGCAGCCTTAAGCA-3′, and probe 5′-TCGCCAGGGACTTTATATTCTGCAAGG-3′. A 25-μL reaction was conducted in a LightCycler (Roche, Indianapolis, IN, USA) with 10 mmol/L of each deoxynucleotide triphosphate, 600 nmol/L of each primer, 200 nmol/L of probe, and 3 μL of RNA. Cycling conditions were 50°C for 30 min and 95°C for 15 min, followed by 45 cycles at 95°C for 15 s and 58°C for 30 s. A quantification curve was constructed with 10-fold serial dilutions of in vitro–transcribed CCHF virus RNA. Positive results were obtained up to a dilution of 10−12, which corresponds to ≈45 virus genome equivalents (geqs) per reaction. Twelve samples had positive results: all 11 samples in group A and 1 in group B (Table). Results for the remaining samples in groups B and C were negative. Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, and a 60-kDa soluble receptor of TNF were previously measured in most of the samples in this study (10), and their values are shown in the Table. Table Epidemiologic, molecular, and clinical data for 12 Albanian patients with suspected Crimean-Congo hemorrhagic fever, 2003–2006* Viral loads ranged from 14 × 106 to 28.99 × 106 geqs/reaction. The highest level was observed in the patient who died (23/03). High loads were observed in all primary case-patients (23/03, 82/03, 178/04, 252/06) except for patient 154/04, from whom a sample was obtained 18 days after onset of disease. All primary case-patients had severe disease with high fever and clinically apparent hemorrhage. All secondary case-patients, except patient 34/03, were contacts of the patient who died (24b/03, husband; 25b/03, brother-in-law; 50/03 and 52/03, cousins; 56/03, sister-in-law; 40/03, son of sister) and had symptoms of disease ≈1 week after the death of patient 23/03. Viral load of secondary case-patients was <250 geqs/reaction, which was much lower than that of primary case-patients. This finding suggests that the disease is more severe in primary case-patients and becomes a milder form in secondary case-patients. Samples of secondary case-patients 24b/03 and 25b/03 were obtained on day 9 of illness, and patient 24b/03 had a 4× higher viral load than patient 25b/03. A possible explanation might be that because patient 24b/03 had closer contact with the person who died, he received a higher dose of virus, which might affect severity of the disease. Other secondary case-patients had milder symptoms with no clinically apparent hemorrhage and were not hospitalized. All hospitalized patients had leukopenia, except for the patient whose sample was taken 18 days after the onset of disease. No correlation was observed between viral load and cytokine levels or platelet counts, which suggests that other factors are involved in pathogenicity and immune response. The real-time RT-PCR was rapid and more sensitive than the RT-nested PCR because 1 additional positive sample was detected. Samples with positive results from the first round of the conventional RT-nested PCR (23/03, 178/04, 252/06) had the highest viral loads when tested by real-time RT-PCR. In conclusion, a 1-step real-time RT-PCR for detection and quantification of CCHF virus was developed, used with clinical samples, and provided informative data on the severity, course, and outcome of CCHF. Further studies, preferably in serial samples of patients, should provide insights into the pathology of CCHF and the effectiveness of antiviral drugs.

Hantaviruses in Serbia and Montenegro.

Hantaviruses are endemic in the Balkan Peninsula. An outbreak of hemorrhagic fever with renal syndrome occurred in 2002 in Serbia and Montenegro. The epidemiologic characteristics and genetic relatedness of Dobrava/Belgrade virus strains responsible for most cases are described.

Antibiotic resistance of Salmonella spp. and Listeria spp. isolates from traditionally made fresh sausages in Greece.

Sixty-five samples of traditionally made fresh sausages obtained from retail shops and butcher shops in northern Greece were screened for the presence of Salmonella spp. and Listeria spp. Salmonella spp. were found in 20% of the samples tested (54% Salmonella typhimurium and 46% Salmonella enteritidis). The prevalence of Listeria spp. in the samples was 26% (12% Listeria monocytogenes, 76% Listeria innocua, and 12% Listeria welshimeri). Nine of 13 Salmonella isolates were found to be resistant to ampicillin and 4 of 13 showed intermediate sensitivity; 1 of 13 was found to be resistant to chloramphenicol and 1 of 13 to tetracycline. Two strains of Salmonella typhimurum were multiresistant (resistant to ampicillin, chloramphenicol, and norfloxacin). All Listeria isolates were sensitive to the antibacterial agents tested that are commonly used for the treatment of human listeriosis.

Suspected Crimean Congo Haemorrhagic Fever cases in Albania

During 2003 to 2006 samples from 34 Albanian patients with suspected Crimean Congo Hemorrhagic Fever (CCHF) were tested by serology and PCR for CCHF virus; negative samples were further tested for hantaviruses, Leptospira spp. and Rickettsia spp. CCHF virus was detected in 38.2% of cases, hantaviruses in 11.7%, and leptospirosis and rickettsiosis were diagnosed in 29.4% and 2.9% of cases, respectively. There is a seasonal and clinical overlapping among the 4 diseases in Albania, suggesting that testing for these agents is necessary in cases with fever and haemorrhagic manifestations.