Anna Papa

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.

Crimean-Congo hemorrhagic fever, southwestern Bulgaria.

To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe multisystem disease characterized by profuse bleeding with a case-fatality rate as high as 30%. The infection is endemic to the Balkans (1,2). In Bulgaria, most cases are reported from the central and eastern parts of the country (3,4). We report a cluster of cases observed in early spring 2008 in southwestern Bulgaria, an area considered at low risk for CCHF outbreaks.

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. n nWe 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. n nOne 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. n nTwelve 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. n n n nTable n nEpidemiologic, molecular, and clinical data for 12 Albanian patients with suspected Crimean-Congo hemorrhagic fever, 2003–2006* n n n nViral 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. n nViral 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. n nThe 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. n nIn 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.

Ecology of the Crimean-Congo hemorrhagic fever endemic area in Albania.

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Albania. Ticks collected from cattle grazing in the endemic areas of Albania were tested for presence of CCHFV RNA, while serum samples collected from goats, cattle, hares, and birds were tested for the presence of specific IgG antibodies to CCHFV. One of the 31 pools prepared, consisting of four female Hyalomma spp. ticks, was found to carry CCHFV RNA with 99.2-100% homology to sequences detected in patients from the same region. Antibodies were not detected in cattle, hares, and birds, but 2/10 goats presented high titers of IgG antibodies. The shepherd of that flock was a member of a family affected by CCHF 10 days before the collection of goats sera, and he presented a mild form of the disease.

HIV-1 subtypes in Yugoslavia.

To gain insight concerning the genetic diversity of HIV-1 viruses associated with the HIV-1 epidemic in Yugoslavia, 45 specimens from HIV-1-infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty-one of 45 specimens (91.2%) were identified as pol subtype B, 2 of 45 as subtype C (4.4%), 1 of 45 as CRF01_AE (2.2%), and 1 as CRF02_AG recombinant (2.2%). Nucleotide divergence among subtype B sequences was 4.8%. Results of this study show that among HIV-1-infected patients in Yugoslavia subtype B predominates (91.5%), whereas non-B subtypes are present at a low percentage, mostly related to travel abroad.

Coronaviruses in Children, Greece

To the Editor: Two recently detected human coronaviruses (HCoVs), NL63 and HKU1, increased the number of coronaviruses known to infect humans to 5 (1–3). HCoV-229E and HCoV-NL63 belong to antigenic group 1, HCoV-OC43 and HCoV-HKU1 belong to antigenic group 2, and severe acute respiratory syndrome (SARS)–associated coronavirus (SARS-CoV) is most closely related to group 2 coronaviruses. In 2005, an optimized pancoronavirus reverse transcription–PCR assay was used to explore the incidence of HCoV-NL63 infection in children in Belgium who had a diagnosis of respiratory tract infection (4). We report the results of an epidemiologic study that used a universal coronavirus RT-PCR assay to detect coronaviruses among children in Greece with acute respiratory tract infections. n nWe tested throat swab specimens obtained from children hospitalized in Greece during June 2003 through May 2004 (200 children 2 months to 14 years of age, mean 4.09 years) and during December 2005 through March 2006 (44 children 1.6–8.5 years of age, mean 5.05). Specimens were obtained the first day of each child’s hospitalization, and all specimens were included in the study, regardless whether other respiratory microorganisms were detected. n nThe 25-μL reaction contained 200 µM dNTPs, 0.2 µM primer PC2S2 (equimolar mixture of 5′-ttatgggttgggattatc-3′ and 5′-tgatgggatgggactatc-3′), 0.8 µM primer PC2As1 (5′-tcatcagaaagaatcatca-3′), 1 μL of enzyme mix from the QIAGEN One-Step RT-PCR Kit (QIAGEN GmbH, Hilden, Germany), and 5 μL of RNA. The initial 30-min reverse transcription step at 48°C was followed by 10 cycles of 20 sec at 94°C, 30 sec at 62°C with a decrease of 1°C per cycle, 40 sec at 72°C; 40 cycles of 20 sec at 94°C, 30 sec at 52°C, 40 sec at 72°C; and a final extension step at 72°C for 10 min. To determine the sensitivity after optimization, we tested quantified RNA in vitro transcripts that included the natural primer binding sites of the respective coronavirus genomes. Sensitivities for SARS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63 were 61.0, 800.0, 8.2, and 82.3 nominal RNA copies per assay, respectively. A separate test was not done for HCoV-HKU1 because it had the same primer binding sites as HCoV-OC43. A phylogenetic tree based on a 400-bp genome fragment of the polymerase gene was constructed (Appendix Figure). n nOf 200 samples collected in 2003–2004, 5 (2.5%) were positive for coronaviruses (2 each for HCoV-NL63 and HCoV-229E and 1 for HCoV-OC43), and of 44 samples collected in 2005–2006, 2 (4.5%) were positive for coronaviruses (1 for HCoV-229E and 1 for HCoV-OC43) (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:EF103180-EF103184,start_term:EF103180,end_term:EF103184,start_term_id:127680602,end_term_id:127680654}}EF103180-EF103184, {type:entrez-nucleotide,attrs:{text:EF394298,term_id:170780329,term_text:EF394298}}EF394298, and {type:entrez-nucleotide,attrs:{text:EF394299,term_id:170780331,term_text:EF394299}}EF394299). CoV-HKU1 was not detected. n nThe amplified genome region is one of the most conserved regions of the coronavirus genome. However, sequences for HCoV-NL63 strains isolated in Greece are genetically closer to the sequence for a strain ({type:entrez-nucleotide,attrs:{text:AY567487,term_id:49035964,term_text:AY567487}}AY567487) isolated in Amsterdam in 2003 (1) than to a strain ({type:entrez-nucleotide,attrs:{text:AY518894,term_id:46369870,term_text:AY518894}}AY518894) from a specimen collected in Rotterdam in 1988 (2) (0.6% vs. 1.1% nucleotide divergence). Sequences for HCoV-229E and HCoV-OC43 strains isolated in Greece differ from sequences for strains isolated elsewhere by 0.5%–1.7%. n nThe HCoV-NL63–positive specimens in our study were obtained from a 9- and a 14-month-old child during winter 2003–2004; no cases were identified during 2005–2006. Specimens positive for HCoV-229E and HCoV-OC43 were detected during both study periods (Table). HCoV-OC43 affected children with a mean age of 3.1 years (median, 1.4 years), and HCoV-229E affected children with a mean (and median) age of 5.5 years. However, no general conclusions can be drawn from these data because number of cases is too few. n n n nTable n nEpidemiologic and laboratory data for patients with coronavirus infection, Greece* n n n nNone of the patients in Greece had an underlying disease, and all recovered completely. Patients infected with HCoV-229E had been hospitalized for upper respiratory tract infections, and those with HCoV-OC43 had lower respiratory tract infections; all cases were mild. Both children infected with HCoV-NL63 had symptoms of lower respiratory tract infections: 1 child had severe pneumonia and was hospitalized for 12 days, while the other had a mild course of bronchiolitis. n nHCoV-NL63 was first identified in Amsterdam, the Netherlands, by van der Hoek et al. (1) from a nasopharyngeal specimen obtained in 2003 from a 7-month-old child with bronchiolitis, conjunctivitis, and fever. One month later, Fouchier et al. (2) reported the characterization of the same virus isolated from a specimen collected in 1988. The specimen had been obtained from an 8-month-old child with pneumonia in Rotterdam, the Netherlands. Later, HCoV-NL63 was detected in 2.5% of bronchiolitis patients <2 years of age in Japan (5) and in most children hospitalized with bronchiolitis in Australia and Canada (6,7). n nCoinfection with HCoVs and other respiratory viruses is frequently observed and is associated with severe clinical syndromes, especially in infants and young children (6,8). Coinfection was observed in 3 of the 7 HCoV-positive patients in our study. The 3 patients were infected with HCoV-OC43 or HCoV-229E; coinfection with respiratory syncytial virus was found in 2 patients, and coinfection with Mycoplasma pneumoniae was found in 1 patient. It was not possible to determine the role of the HCoVs in these coinfections. In addition, because coronaviruses can be detected even 3 weeks after an acute episode, some cases of coinfection might represent former rather than current HCoV infection (9). n nIn conclusion, we detected 3 types of HCoVs in Greece: 229E, OC43, and NL63. This finding provides initial insight into the epidemiologic features of coronaviruses in Greece. Further studies are needed to find the exact clinical effect of these HCoVs in humans and to elucidate the epidemiology of coronaviruses worldwide.