Viral load as predictor of crimean-congo hemorrhagic fever outcome.
We used quantitative real-time reverse transcription-PCR to measure viral load viral load
The concentration of a virus, such as HIV, in the blood.
n a measure of the number of virus particles present in the bloodstream, expressed as copies per milliliter. in serum from 24 patients in Kosovo who had acute 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. . Viral load correlated with clinical disease and antibodies and could be used as a predictor of disease outcome.
Crimean-Congo hemorrhagic fever (CCHF CCHF Crimean-Congo Hemorrhagic Fever
CCHF Congo Cerebral Hemorrhage Fever ), caused by CCHF virus, is a potentially fatal infection in Africa, Asia, Eastern Europe, and the Middle East. CCHF virus is transmitted to humans by bites of Ixodid ticks and from person to person by contact with blood or blood-containing body fluids. Therefore, nosocomial nosocomial /noso·co·mi·al/ (nos?o-ko´me-il) pertaining to or originating in a hospital.
1. Of or relating to a hospital.
2. and intrafamiliar cases are frequently reported in CCHF outbreaks (1). CCHF can be treated with ribavirin ribavirin /ri·ba·vi·rin/ (ri?bah-vi´rin) a broad-spectrum antiviral used in the treatment of severe viral pneumonia caused by respiratory syncytial virus, particularly in high-risk infants; also used in conjunction with interferon , but the decision about which CCHF patients should be given the drug may be difficult (2,3). Classification of patients according to criteria of disease severity is an important step in deciding whether to initiate antiviral therapy and is based on clinical data and biochemical test results (2,4). Because of severe side effects Side effects
Effects of a proposed project on other parts of the firm. from ribavirin treatment, early laboratory confirmation would be desirable to establish stronger criteria for case classification of CCHF.
Real-time PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) has enabled measurement of viral loads, monitoring of antiviral treatment effects and emergence of antiviral resistant strains, and prediction of disease progression and outcome (5). For many viral diseases, including hemorrhagic fevers Hemorrhagic Fevers Definition
Hemorrhagic fevers are caused by viruses that exist throughout the world. However, they are most common in tropical areas. , viral load measurement has become an integral part of disease management (6-9). However, to our knowledge, the usefulness of viral load monitoring for CCHF has never been investigated. Given the importance of predicting CCHF disease severity and risk for death, our aim was to measure viral load in CCHF patients and to correlate it with other laboratory parameters and disease outcomes.
Serum samples were obtained from CCHF patients from Kosovo in 2001 (www.who.int/csr/don/2001_06_29e/en), 2003, and 2005. Clinical and biochemical data were provided by the Clinic of Infectious Diseases, Pristina, Kosovo. Presence of an acute febrile febrile /feb·rile/ (feb´ril) pertaining to or characterized by fever.
Of, relating to, or characterized by fever; feverish. syndrome characterized by malaise, nausea, fever, and bleeding from various sites was reported as well as possible modes of infection. Leukocyte leukocyte (l`kəsīt'): see blood.
or white blood cell or white corpuscle and platelet counts, aspartate aspartate /as·par·tate/ (ah-spahr´tat) a salt of aspartic acid, or aspartic acid in dissociated form.
1. A salt of aspartic acid.
2. and alanine aminotransferase alanine aminotransferase /al·a·nine ami·no·trans·fer·ase/ (ah-me?no-trans´fer-as) alanine transaminase.
n. Abbr. ALT
See SGPT. levels, activated partial thromboplastin times, and creatinine values were available for most patients. Patients were categorized into 3 groups according to disease severity: fatal, severe, or moderate cases. On the basis of classification by Swanepoel (4), severe cases were defined by the presence of hemorrhagic Hemorrhagic
A condition resulting in massive, difficult-to-control bleeding.
Mentioned in: Hantavirus Infections
pertaining to or characterized by hemorrhage. manifestations (epistaxis epistaxis /ep·i·stax·is/ (-stak´sis) nosebleed; hemorrhage from the nose, usually due to rupture of small vessels overlying the anterior part of the cartilaginous nasal septum.
n. , hematemesis hematemesis /he·ma·tem·e·sis/ (he?mah-tem´e-sis) the vomiting of blood.
The vomiting of blood. , and melena melena /me·le·na/ (me-le´nah) the passage of dark stools stained with altered blood.
n. ), lowered blood pressure (<100/60 mm Hg), and raised serum creatinine and transaminase transaminase /trans·am·i·nase/ (-am´i-nas) aminotransferase.
See aminotransferase. levels.
Serologic testing for anti-CCHF virus immunoglobulin (Ig) M and IgG was done by ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. . Molecular data were obtained by real-time reverse transcription--PCR (RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. ) with a limit of detection of 240 copies/mL of sample as recently described (10). Some assay modifications were necessary for accurate quantitation of viral load. Synthetic RNA RNA: see nucleic acid.
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 was generated as a quantitative calibrator calibrator
an instrument for dilating a tubular structure or for determining the caliber of such a structure. , and a competitive internal control was constructed as previously described to detect possible influences of PCR inhibitors (11). The original real-time RT-PCR protocol was complemented by the addition of 200 pmol/[micro]L of internal control probe YFP YFP Yellow Fluorescent Protein (microscopy)
YFP Floating Power Barge (Non Self-Propelled)
YFP Your Favorite Protein 2 (5'-ROX-ATCGTTCGTTGAGCGATTAG-CAG-BBQ-3'). This probe recognizes an alternative binding site introduced in the target gene by overlap-extension PCR (11). The standard curve for CCHF virus quantitation was based on synthetic calibrator RNA with concentrations from 24 x [10.sup.5] to 24 x [10.sup.1] copies/mL. Statistical analysis was performed with statistical software R version 2.2.1 (www.r-project.org) and the Statgraphics 5 package (Manugistics, Dresden, Germany).
A total of 24 patients had clinical, 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. , and molecular confirmation of CCHF (Table). All 24 patients had an acute febrile syndrome, 8 reported tick bites, and 2 had been exposed in a hospital. For the 9 patients who died, only 1 serum sample was available from each. For the 9 patients with severe disease and the 6 with moderate disease, 2-3 consecutive samples were available.
From the 24 patients, 43 serum samples were tested by real-time RT-PCR and ELISA (Table). Viral loads ranged from [10.sup.2] to [10.sup.10] copies per milliliter milliliter /mil·li·li·ter/ (mL) (-le?ter) one thousandth (10-3) of a liter.
n. Abbr. of serum, depending on the day of illness, the severity of disease, and the results of serologic analyses (Table). Whether early laboratory findings could serve as prognostic markers for outcome was explored. Because prognostic information is most relevant in the first week of disease, only samples taken up to day 7 of symptoms from any patient were included in the analysis. Average sampling days did not differ between patients who died and those who survived (5.0 and 3.9 days, respectively, p = 0.28, analysis of variance [ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there ]). No patient from either group had detectable IgG in the first week. Although we suspected that early development of IgM might correlate with good outcome, such correlation was not found. Of those patients in whom IgM was detected up to day 7, 5 (62%) died. Of those without IgM in the first week, 3 (42%) died (insignificant difference in 1-way ANOVA, p = 0.6). Furthermore, the presence of IgM in the first week did not correlate with viral load, which suggests that virus levels in the first week can be regarded as an independent prognostic parameter. Namely, viral load seemed to be strongly related to the clinical classification (p [less than or equal to] 0.001), with the average log value 9.25 (1.78 x [10.sup.9]) in the group of patients who died and 6.91 (8.06 x [10.sup.6]) in the group who survived (Figure).
To determine whether IgG could influence viremia viremia /vi·re·mia/ (vi-re´me-ah) the presence of viruses in the blood.
The presence of viruses in the bloodstream. , viral loads were correlated with log-transformed reciprocal antibody titers. Quantitative IgG levels showed a highly significant inverse correlation with viral loads (p<0.0001) (Figure). It was thus reasoned that IgG levels could influence the later course of disease and, in particular, could reflect a discrimination between severe and moderate cases. In samples from both categories, no relationship between IgG and clinical classification (p = 0.65) was determined after day 7. Also, no significant relationship was found between clinical classification and viral load (p = 0.74) in severe versus moderate cases. On average, viral load log value was 2.38 in severe cases and 2.69 in moderate cases (Figure).
This study describes the differential influences of CCHF viral load, IgM, IgG, and clinical outcome. CCHF viral load, but not IgM, could be used as a predictor of CCHF outcome. It was unexpected that IgM correlated with neither outcome nor viral load. On the contrary, quantitative IgG levels inversely correlated with viral loads, which suggests that IgG might neutralize virus in vivo. The fact that virus titers decreased in survivors independent of antibodies during the first week implies involvement of innate or cellular immune mechanisms in the elimination of CCHF virus.
CCHF viral load ranged from 102 to [10.sup.10] copies/mL in the serum samples. It was shown that viral load of>[10.sup.8] copies/mL is a strong factor (p<0.001) for differentiating CCHF patients who died from those who survived. However, viral load does not help differentiate between severe and moderate cases according to common case definitions (4). The same was true for IgM levels. Viral load is also useful for estimating need for infection control measures. Viral loads measured in our patients were high, > [10.sup.9] copies/mL, higher than viral loads in other arboviral diseases that are not easily transmitted in the hospital, e.g., dengue dengue
or breakbone fever or dandy fever
Infectious, disabling mosquito-borne fever. Other symptoms include extreme joint pain and stiffness, intense pain behind the eyes, a return of fever after brief pause, and a characteristic rash. (12). This finding could help explain why CCHF virus causes nosocomial infections on a regular basis. Another use for this finding is systematic monitoring of patients receiving ribavirin therapy. In the absence of sufficiently large numbers of treated patients, however, we could not investigate this application.
We thank Maja Pohar for statistical analysis.
This work was partially supported by RiViGene (contract no. SSPE-CT-2005-022639).
Dr Duh duh
Used to express disdain for something deemed stupid or obvious, especially a self-evident remark.
[Imitative of an utterance attributed to slow-witted people.] is a microbiologist at the Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia. She works with emerging and reemerging tickborne pathogens and their ecology, molecular epidemiology, and diagnosis.
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Address for correspondence: Tatjana Avsic-Zupanc, Institute of Microbiology and Immunology, Faculty of Medicine/University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia; email: email@example.com
Darja Duh, * Ana Saksida, * Miroslav Petrovec, * Salih Ahmeti, ([dagger]) lusuf Dedushaj, ([double dagger]) Marcus Panning, [section] Christian Drosten, [section] and Tatjana Avic-Zupanc *
* Institute of Microbiology and Immunology, Ljubljana, Slovenia; ([dagger]) Clinic of Infectious Diseases, Pristina, Kosovo; ([double dagger]) National Institute of Public Health, Pristina, Kosovo; and [section] Bernhard Nocht Institute for Tropical Medicine Bernhard Nocht Institute for Tropical Medicine; (Bernhard-Nocht-Institut für Tropenmedizin) is a medical institution based in Hamburg, Germany which is dedicated to research, treatment, training and therapy of tropical and infectious diseases, (including HIV). , Hamburg, Germany
Table. Detection of CCHF viral load by real-time RT-PCR in serum of patients with acute CCHF, Kosovo * Disease Day of Patient no. severity illness IgM titer IgG titer 1 Fatal 2 Neg Neg 2 Fatal 3 Neg Neg 3 Fatal 4 Neg Neg 4 Fatal 9 Neg Neg 5 Fatal 4 1,600 Neg 6 Fatal 6 3,200 Neg 7 Fatal 7 400 Neg 8 Fatal 7 800 Neg 9 Fatal 7 800 Neg 10 Severe 8 >6,400 Neg 18 >6,400 400 11 Severe 9 Neg Neg 24 >6,400 >6,400 12 Severe 4 800 Neg 16 >6,400 3,200 13 Severe 2 Neg Neg 9 >6,400 400 42 >6,400 3,200 14 Severe 8 Neg Neg 14 6,400 Neg 15 Severe 3 Neg Neg 14 6,400 200 16 Severe 3 >1,600 Neg 6 >800 >800 17 Severe 4 Neg Neg 10 >6,400 100 18 Severe 2 Neg Neg 9 6,400 400 23 >6,400 >6,400 19 Moderate 12 >6,400 Neg 17 >6,400 800 20 Moderate 9 >6,400 1,600 11 >6,400 3,200 21 Moderate 12 >6,400 800 13 >6,400 3,200 32 >6,400 >6,400 22 Moderate 10 >6,400 Neg 19 >6,400 >6,400 26 >6,400 >6,400 23 Moderate 9 6,400 6,400 20 >6,400 >6,400 24 Moderate 7 400 Neg 18 >6,400 6 4nn Patient no. Viral load (copies/mL) 1 6.5100 x [10.sup.8] 2 2.5040 x [10.sup.9] 3 2.7400 x [10.sup.9] 4 3.3840 x [10.sup.9] 5 1.0160 x [10.sup.8] 6 1.3450 x [10.sup.10] 7 1.8675 x [10.sup.9] 8 3.4800 x [10.sup.9] 9 1.2920 x [10.sup.9] 10 1.6050 x [10.sup.6] 1.1500 x [10.sup.5] 11 2.3250 x [10.sup.7] Neg 12 3.3900 x [10.sup.6] Neg 13 1.0430 x [10.sup.9] 3.1200 x [10.sup.3] Neg 14 8.1000 x [10.sup.6] 2.0100 x [10.sup.3] 15 3.8100 x [10.sup.7] Neg 16 2.5235 x [10.sup.6] 2.2350 x [10.sup.4] 17 3.3600 x [10.sup.7] 3.8100 x [10.sup.4] 18 7.8500 x [10.sup.7] 3.2000 x [10.sup.7] Neg 19 5.7525 x [10.sup.4] Neg 20 1.9600 x [10.sup.3] Neg 21 1.9191 x [10.sup.5] 1.0240 x [10.sup.4] Neg 22 4.6400 x [10.sup.4] 1.2000 x [10.sup.3] Neg 23 7.6800 x [10.sup.3] 7.5000 x [10.sup.2] 24 7.4400 x [10.sup.5] Neg * CCHF, Crimean-Congo hemorrhagic fever: RT-PCR, reverse transcription-PCR; Ig, immunoglobulin; neg, negative result.