[The Importance of Differential Diagnosis During Pandemic: A Case Report with Coexistence of COVID-19, Brucellosis and Crimean-Congo Hemorrhagic Fever]. / Pandemi Döneminde Ayirici Taninin Önemi: COVID-19, Bruselloz ve Kirim-Kongo Kanamali Atesi Birlikteligi Olan Bir Olgu Sunumu.

The coronavirus disease-2019 (COVID-19) pandemic, which affects millions of people around the world, has been affecting our country since March 2020. The fact that the symptoms such as fever, myalgia, headache, joint pain which are common in COVID-19 patients are quite similar to the symptoms of diseases such as Crimean-Congo hemorrhagic fever (CCHF) and Brucellosis. This may cause a diagnostic confusion in regions where these diseases are seen as endemic. In this report, a patient hospitalized with a pre-diagnosis of COVID-19 and diagnosed with acute Brucellosis, CCHF and COVID-19 during followup was presented. A 31-year-old female patient living in a rural area admitted to the emergency service with complaints of fever, weakness, headache, and body/joint pain. Physical examination revealed a temperature of 38.3°C, a pulse rate of 102/minute, and a peripheral capillary oxygen saturation of 97% in room air. The system examination was normal. In the laboratory findings, an increase in liver enzymes and acute phase reactants was observed and the platelet count was at the lower limit of the normal range. In terms of COVID-19, no involvement compatible with COVID-19 was detected in the thorax computed tomography (CT) of the patient whose nasopharyngeal and oropharyngeal mixed swab samples were taken.The patient was transferred to our infectious diseases service with a pre-diagnosis of COVID-19 and CCHF. Serum samples were sent to the Public Health Agency Microbiology Reference Laboratory Department (PHA-MRLD) for CCHF diagnostic tests and supportive treatment was started. Brucella Rose Bengal and Coombs' immuncapture (1/1280 titer) tests were found as positive in the patient, who was examined for brucellosis because of living in a rural area and having a history of consuming fresh dairy products. In the tests performed at PHA-MRLD, CCHF-specific IgM positivity and the presence of viral RNA were detected. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) reverse-transcriptase polymerase chain reaction (RT-PCR) test was negative. For Brucellosis, doxycycline and rifampicin were added to the treatment of the patient whom was given supportive therapy for CCHF. In the followup, the patient's fever was persisting and loss of taste and smell complaint developed. In this context, COVID-19 test was repeated and resulted as positive. Upon this, hydroxychloroquine sulfate treatment was started due to the recommendation of the current Ministry of Health Scientific Committee Guide. No new infiltration was detected in the chest radiography of the patient. The patient's fever subsided during follow-up and laboratory findings improved. The treatment of brucellosis was completed to eight weeks at the outpatient clinic. No problems were detected in the follow-up. This report was prepared because of a case with simultaneous brucellosis, CCHF and COVID-19 infections which could not be encountered in the literature review. As a result; in regions such as our country where both brucellosis and CCHF are seen as endemic, it is very important to keep these diseases in mind in the differential diagnosis of COVID-19 infection.

Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.

CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/µl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.

Multi-omics insights into host-viral response and pathogenesis in Crimean-Congo hemorrhagic fever viruses for novel therapeutic target.

The pathogenesis and host-viral interactions of the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are convoluted and not well evaluated. Application of the multi-omics system biology approaches, including biological network analysis in elucidating the complex host-viral response, interrogates the viral pathogenesis. The present study aimed to fingerprint the system-level alterations during acute CCHFV-infection and the cellular immune responses during productive CCHFV-replication in vitro. We used system-wide network-based system biology analysis of peripheral blood mononuclear cells (PBMCs) from a longitudinal cohort of CCHF patients during the acute phase of infection and after one year of recovery (convalescent phase) followed by untargeted quantitative proteomics analysis of the most permissive CCHFV-infected Huh7 and SW13 cells. In the RNAseq analysis of the PBMCs, comparing the acute and convalescent-phase, we observed system-level host's metabolic reprogramming towards central carbon and energy metabolism (CCEM) with distinct upregulation of oxidative phosphorylation (OXPHOS) during CCHFV-infection. Upon application of network-based system biology methods, negative coordination of the biological signaling systems like FOXO/Notch axis and Akt/mTOR/HIF-1 signaling with metabolic pathways during CCHFV-infection were observed. The temporal quantitative proteomics in Huh7 showed a dynamic change in the CCEM over time and concordant with the cross-sectional proteomics in SW13 cells. By blocking the two key CCEM pathways, glycolysis and glutaminolysis, viral replication was inhibited in vitro. Activation of key interferon stimulating genes during infection suggested the role of type I and II interferon-mediated antiviral mechanisms both at the system level and during progressive replication.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging disease that is increasingly spreading to new populations. The condition is now endemic in almost 30 countries in sub-Saharan Africa, South-Eastern Europe, the Middle East and Central Asia. CCHF is caused by a tick-borne virus and can cause uncontrolled bleeding. It has a mortality rate of up to 40%, and there are currently no vaccines or effective treatments available. All viruses depend entirely on their hosts for reproduction, and they achieve this through hijacking the molecular machinery of the cells they infect. However, little is known about how the CCHF virus does this and how the cells respond. To understand more about the relationship between the cell's metabolism and viral replication, Neogi, Elaldi et al. studied immune cells taken from patients during an infection and one year later. The gene activity of the cells showed that the virus prefers to hijack processes known as central carbon and energy metabolism. These are the main regulator of the cellular energy supply and the production of essential chemicals. By using cancer drugs to block these key pathways, Neogi, Elaldi et al. could reduce the viral reproduction in laboratory cells. These findings provide a clearer understanding of how the CCHF virus replicates inside human cells. By interfering with these processes, researchers could develop new antiviral strategies to treat the disease. One of the cancer drugs tested in cells, 2-DG, has been approved for emergency use against COVID-19 in some countries. Neogi, Elaldi et al. are now studying this further in animals with the hope of reaching clinical trials in the future.