Viral load and Crimean-Congo hemorrhagic fever.To the Editor: Crimean-Congo hemorrhagic fever Crimean-Congo hemorrhagic fever a zoonotic disease of humans, in central Asia through to eastern Europe, who are in contact with livestock. Caused by a bunyavirus, it is transmitted by ticks. The principal signs are fever, widespread hemorrhages and necrotizing hepatitis. (CCHF CCHF Crimean-Congo Hemorrhagic Fever CCHF Congo Cerebral Hemorrhage Fever ) is a severe viral disease transmitted to humans by tick bite or contact with blood, excreta excreta /ex·cre·ta/ (eks-kret´ah) excretion (2). ex·cre·ta pl.n. Waste matter, such as sweat or feces, discharged from the body. , 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 Balkan Peninsula, southeasternmost peninsula of Europe, c.200,000 sq mi (518,000 sq km), bounded by the Black Sea, Sea of Marmara, Aegean Sea, Mediterranean Sea, Ionian Sea, and Adriatic Sea. (1-5). Prompt diagnosis of the disease is essential for preventing human-to-human transmission. Reverse transcription-PCR (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) 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 RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic 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 viral load n. The concentration of a virus, such as HIV, in the blood. viral load, n a measure of the number of virus particles present in the bloodstream, expressed as copies per milliliter. 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 PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) (9). Group B contained samples from 5 patients who had negative RT-nested PCR results and positive serologic se·rol·o·gy n. pl. se·rol·o·gies 1. The science that deals with the properties and reactions of serums, especially blood serum. 2. 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-[micro]L reaction was conducted in a LightCycler (Roche, Indianapolis, IN, USA) with 10 mmol/L of each deoxynucleotide triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals. tri·phos·phate n. A salt or ester containing three phosphate groups. , 600 nmol/L of each primer, 200 nmol/L of probe, and 3 [micro]L of RNA. Cycling conditions were 50[degrees]C for 30 min and 95[degrees]C for 15 min, followed by 45 cycles at 95[degrees]C for 15 s and 58[degrees]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.sup.-2], which corresponds to [approximately equal 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 tumor necrosis Death of tumor tissue, a common event in aggressive CAs in which the tumor rapidly outgrows its blood supply, resulting in tumor cell death. Cf Apoptosis. factor-[alpha] (TNF-[alpha]), interleukin-6 (IL-6), IL-10, and a 60-kDa soluble receptor of TNF TNF abbr. tumor necrosis factor TNF, n an abbreviation for tumor necrosis f were previously measured in most of the samples in this study (10), and their values are shown in the Table. Viral loads ranged from 14 x [10.sup.6] to 28.99 x [10.sup.6] 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 [approximately equal to] 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 4x 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 leukopenia /leu·ko·pe·nia/ (-pe´ne-ah) reduction of the number of leukocytes in the blood below about 5000 per cubic mm.leukope´nic basophilic leukopenia basophilopenia. , except for the patient whose sample was taken 18 days after the onset of disease. No correlation was observed between viral load and cytokine Cytokine Any of a group of soluble proteins that are released by a cell to send messages which are delivered to the same cell (autocrine), an adjacent cell (paracrine), or a distant cell (endocrine). levels or platelet counts, which suggests that other factors are involved in pathogenicity and immune response immune response n. An integrated bodily response to an antigen, especially one mediated by lymphocytes and involving recognition of antigens by specific antibodies or previously sensitized lymphocytes. . 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 Antiviral Drugs Definition Antiviral drugs are medicines that cure or control virus infections. Purpose Antivirals are used to treat infections caused by viruses. . References (1.) Papa A, Bino S, Llagami A, Brahimaj B, Papadimitriou E, Pavlidou V, et al. Crimean-Congo hemorrhagic fever in Albania, 2001. Eur J Clin Microbiol Infect Dis. 2002;21:603-6. (2.) Papa A, Bozovic B, Pavlidou V, Papadimitriou E, Pelemis M, Antoniadis A. Genetic detection and isolation of Crimean-Congo hemorrhagic fever virus, Kosovo, Yugoslavia. Emerg Infect Dis. 2002;8:852-4. (3.) Papa A, Christova I, Papadimitriou E, Antoniadis A. Crimean-Congo hemorrhagic fever in Bulgaria. Emerg Infect Dis. 2004; 10:1465-7. (4.) Ahmeti S, Raka L. Crimean-Congo haemorrhagic fever Noun 1. haemorrhagic fever - a group of illnesses caused by a viral infection (usually restricted to a specific geographic area); fever and gastrointestinal symptoms are followed by capillary hemorrhage in Kosova: a fatal case report. Virol J. 2006;3:85. (5.) Drosten C, Minnak D, Emmerich P, Schmitz H, Reinicke T. Crimean-Congo hemorrhagic fever in Kosovo. J Clin Microbiol. 2002;40:1122-3. (6.) Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H, et al. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus Lassa virus n. A virus of the genus Arenavirus that causes Lassa fever. , Crimean-Congo hemorrhagic fever virus, Rift Valley fever Rift Valley fever An arthropod-borne (primarily mosquito), acute, febrile, viral disease of humans and numerous species of animals. Rift Valley fever is caused by a ribonucleic acid (RNA) virus in the genus Phlebovirus of the family Bunyaviridae. virus, dengue virus dengue virus n. A virus of the genus Flavivirus that is the cause of dengue. , and yellow fever virus yellow fever virus n. An arbovirus of the genus Flavivirus that causes yellow fever and is transmitted by mosquitoes. by real-time reverse transcription-PCR. J Clin Microbiol. 2002;40:2323-30. (7.) Duh duh interj. Used to express disdain for something deemed stupid or obvious, especially a self-evident remark. [Imitative of an utterance attributed to slow-witted people.] D, Saksida A, Petrovec M, Dedushaj I, Avsic-Zupanc T. Novel one-step realtime RT-PCR assay for rapid and specific diagnosis of Crimean-Congo hemorrhagic fever encountered in the Balkans. J Virol Methods. 2006; 133:175-9. (8.) Yapar M, Aydogan H, Pahsa A, Besirbellioglu A, Bodur H, Basustaoglu AC, et al. Rapid and quantitative detection of Crimean-Congo hemorrhagic fever virus by one-step real-time reverse transcriptase-PCR. Jpn J lnfect Dis. 2005;58:358-62. (9.) Schwarz TF, Nsanze H, Longson M, Nitschko H, Gilch S, Shurie H, et al. Polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is for diagnosis and identification of distinct variants of Crimean-Congo hemorrhagic fever virus in the United Arab Emirates United Arab Emirates, federation of sheikhdoms (2005 est. pop. 2,563,000), c.30,000 sq mi (77,700 sq km), SE Arabia, on the Persian Gulf and the Gulf of Oman. . Am J Trop Med Hyg. 1996;55:190-6. (10.) Papa A, Bino S, Velo E, Harxhi A, Kota M, Antoniadis A. Cytokine levels in Crimean-Congo Hemorrhagic Fever. J Clin Virol. 2006;36:272-6. Address for correspondence: Anna Papa, Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki The Aristotle University of Thessaloniki (often referred to in English as Aristotelian University), named after the philosopher Aristotle, is the largest university of Greece. Its campus covers 429 metric acres close to the center of the city of Thessaloniki. , 54124 Thessaloniki, Greece; email: annap@ med.auth.gr Anna Papa, * Christian Drosten, ([dagger]) Silvia Bino, ([double dagger double dagger n. A reference mark (  ) used in printing and writing. Also called diesis.Noun 1. ]) Evangelia Papadimitriou, * Marcus Panning, ([dagger]) Enkelejda Velo, ([double dagger]) Majlinda Kota, ([double dagger]) Arjan Harxhi, ([section]) and Antonis Antoniadis * * Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece; ([dagger]) Berhard Nocht Institute for Tropical Medicine tropical medicine, study, diagnosis, treatment, and prevention of certain diseases prevalent in the tropics. The warmth and humidity of the tropics and the often unsanitary conditions under which so many people in those areas live contribute to the development and , Hamburg, Germany; ([double dagger]) Institute of Public Health, Tirana, Albania; and ([section]) University Hospital, Tirana, Albania
Table. Epidemiologic, molecular, and clinical data for 12 Albanian
patients with suspected Crimean-Congo hemorrhagic fever, 2003-2006 *
Day of In
Patient illness hospital Outcome
Group A primary
23/03 6 Yes D
82/03 5 Yes R
154/04 18 Yes R
178/04 4 Yes R
252/06 2 Yes R
Group A secondary
24b/03 9 Yes R
25b/03 9 Yes R
50/03 3 No R
52/03 3 No R
56/03 5 No R
34/03 5 Yes R
Group B secondary
40/03 7 No R
Real-time
Patient RT-PCR, TNF-[alpha],
gegs/reaction IFA pg/mL
Group A primary
23/03 28,990,000 + 68.5
82/03 450 + N
154/04 33 + ND
178/04 7,271,000 +
252/06 4049 - ND
Group A secondary
24b/03 166 + 1,445
25b/03 40 + N
50/03 240 - N
52/03 18 - N
56/03 62 + N
34/03 46 - N
Group B secondary
40/03 14 + N
sTNF-R, IL-6, IL-10,
Patient ng/mL pg/mL pg/mL
Group A primary
23/03 14.0 109.7 388.3
82/03 N 17.0 23.9
154/04 ND ND ND
178/04 ND ND ND
252/06 ND ND ND
Group A secondary
24b/03 N 114.2 N
25b/03 N 10.3 N
50/03 N N 43.4
52/03 N N 9.9
56/03 N 26.1 N
34/03 8.9 N N
Group B secondary
40/03 N N 23.1
Leukocytes, Platelets,
Patient x [10.sup.9]/L x [10.sup.9]/L
Group A primary
23/03 1,700 36,200
82/03 2,300 96,830
154/04 15,000 71,400
178/04 4,100 62,550
252/06 3,700 117,900
Group A secondary
24b/03 4,800 63,800
25b/03 3,800 63,000
50/03 ND ND
52/03 ND ND
56/03 ND ND
34/03 8,000 102,000
Group B secondary
40/03 ND ND
* RT-PCR, reverse transcription-PCR; geqs, genome equivalents;
IFA, immunofluorescent assay; TNF-[alpha], tumor necrosis
factor-[alpha]; sTNF-R, soluble TNF-[alpha] receptor; IL-6,
interleukin-6; D, died; +, positive; R, recovered; N, normal
value; ND, not done; -, negative.
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