Giriş J Arthropod-Borne Dis, December 2017, 11(4): 463–468 F Duygu et al.: Brucellosis in … 463 http://jad.tums.ac.ir Published Online: December 30, 2017 Original Article Brucellosis in Patients with Crimean-Congo Hemorrhagic Fever Fazilet Duygu 1, *Tugba Sari 1, Turan Kaya 2, Nermin Bulut 3 1Public Health Institution of Turkey, Ankara, Turkey 2Tokat State Hospital, Department of Infectious Diseases and Clinical Microbiology, Tokat, Turkey 3Gaziosmanpasa University, Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Tokat, Turkey (Received 15 May 2016; accepted 3 Dec 2017) Abstract Background: Crimean-Congo hemorrhagic fever (CCHF) is a fatal zoonotic viral disease caused by infection with a tick-borne virus of the genus Nairovirus. In this study, we investigated the incidence of brucellosis in patients diag- nosed with CCHF. Methods: Overall, 169 patients hospitalized with an initial diagnosis of CCHF were included in 2011 in Tokat/ Turkey. Immunoglobulin M (IgM) antibodies and/or PCR results were used in the laboratory diagnosis of CCHF, while plate and standard tube agglutination (STA) tests were used to diagnose brucellosis. Results: Overall, 120 patients (79%) with positive PCR tests were diagnosed with CCHF. Five (4.16%) were also diagnosed with brucellosis based on the positive plate and STA test results. Four patients (2.36%) had negative CCHF PCR and positive STA test results. Conclusion: Brucellosis and CCHF can mimic each other and that all patients with CCHF or brucellosis should be screened for both conditions. Keywords: Crimean-Congo hemorrhagic fever, Haemorrhagic fever, Brucellosis, Zoonosis Introduction Crimean-Congo hemorrhagic fever (CCHF) is a viral disease occurring in differ- ent regions worldwide and may be life threat- ening due to the manifestations of fever ac- companied by hemorrhage. It is a zoonosis caused by Nairoviruses transmitted via Hy- alomma ticks (1, 2). This disease, observed in more than 30 countries, including coun- tries in Asia, Europe, and Africa, results in a mortality rate of 5.4–80% (3, 4). Tokat Prov- ince in northern Turkey is in an endemic area for CCHF, which is also endemic in the middle and eastern parts of the country. CCHF is characterized by fever, malaise, sweating, anorexia, and arthralgia, and vari- ous nonspecific symptoms following an in- cubation period lasting less than a week. As the disease progresses, it may potentially man- ifest disseminated intravascular coagulation (DIC) and shock (5). Brucellosis caused by Brucella bacteria represents a zoonosis transmitted to humans via the body fluids and secretions of animals such as sheep, goats, cows, buffalos, and pigs and via dairy products made with contaminated milk. After a 2–3 week incubation period, non- specific symptoms that include fatigue, lack of appetite, muscle and joint pain, and subfe- brile fever may occur. Depending on the part of the body affected by the infection, different clinical characteristics may manifest (6, 7). As the symptoms of brucellosis are non- specific, differential diagnoses are difficult and misdiagnoses are frequent. This trial de- signed to assess the incidence of brucellosis among patients with a preliminary diagnosis of CCHF and the clinical conditions resulting from the co-existence of the two zoonoses. *Corresponding author: Dr Tugba Sari, E-mail: drtugba82@gmail.com J Arthropod-Borne Dis, December 2017, 11(4): 463–468 F Duygu et al.: Brucellosis in … 464 http://jad.tums.ac.ir Published Online: December 30, 2017 Materials and Methods This is a prospective cohort study imple- mented from Apr 2011 and Jul 2011 in To- kat, Turkey. One hundred and sixty-nine in- patients monitored at the Tokat State Hospi- tal with a preliminary diagnosis of CCHF were enrolled in the trial. All the biochemical anal- yses were performed by auto-analyzer and complete blood counts were performed by automatic hemocounter at central laboratory of our hospital. The Public Health Institution of Turkey routinely submitted case definition forms and serum samples obtained from the suspected cases (the first sample) to the National Ref- erence Laboratory. All patients preliminarily diagnosed with CCHF based on the case definition criteria were hospitalized and followed up, as re- quired by the regulations. Diagnosis of CCHF and brucellosis requires satisfaction of the following criteria 1) Compatible clinical pic- ture, 2) Diagnosis of CCHF based on detection of CCHF IgM antibodies by ELISA and/or CCHFV RNA with rreverse transcription pol- ymerase chain reaction assays and direct se- quence analyses (8, 3), 3). All patients un- derwent a Brucella Rose-Bengal slide agglu- tination test, with blood cultures performed for patients with a positive result based on a standard tube agglutination (STA) test and/ or Coombs’ tests (titers≥1/160) (9, 10). We used a commercial kit (Cromatest, Knicker- bocker Laboratories, Barcelona, Spain) for the SAT. The Coombs’ test used anti-human gamma globulin sera (Ortho Diagnostic Sys- tems, Madrid, Spain) to detect blocking an- tibodies. In cases of positive sera, we per- formed serial tube dilutions that ranged from 1:10 to 1:1240. The BACTEC 9050 blood culture system (Becton, Dickinson and Com- pany, USA) was used to culture Brucella. The histories and physical examination results of the patients diagnosed with brucellosis based on the clinical and laboratory findings were recorded. The age, gender, white blood cell and platelet counts, and the aspartate aminotrans- ferase (AST) and alanine aminotransferase (ALT) levels of the patients were recorded. Statistical analyses were conducted using SPSS ver. 17.0 for Windows (Inc., Chicago, IL, USA). Qualitative data were expressed as number (percentage) and mean comparisons for continuous variables were performed us- ing independent-group t-tests. Results Ninety patients (53.2%) were men, and the mean age of the patients was 32.8±9.21yr. The Brucella STA test was positive for nine of the 169 patients (5.02%). These nine pa- tients were living in rural areas. They all had a history of stockbreeding and consumption of unpasteurized milk products. None had a history of brucellosis. One hundred and twenty patients with positive CCHF PCR results were diagnosed with CCHF (79%). Five of those diagnosed with CCHF also had positive Brucella slide and tube agglutination test results (4.16%). All patients had general symptoms of in- fection and fever (100%). One of the pa- tients had sacroiliitis (11.1%) and two had splenomegaly (22.2%). Four of the 169 pa- tients hospitalized with an initial diagnosis of CCHF was found to be CCHF PCR nega- tive and Brucella STA positive (2.36%). All patients had thrombocytopenia and leukope- nia. Two patients had anemia. Five patients had elevated ALT levels and eight patients had elevated AST levels. The demographic characteristics and laboratory results of the patients are presented in Table 1. J Arthropod-Borne Dis, December 2017, 11(4): 463–468 F Duygu et al.: Brucellosis in … 465 http://jad.tums.ac.ir Published Online: December 30, 2017 Table 1. Demographic characteristics and laboratory results of the patients diagnosed with brucellosis Patient number 1 2 3 4 5 6 7 8 9 Age (yr) 62 23 35 46 22 42 40 46 52 Gender Male Male Male Male Female Male Female Female Male Living area of the patients Rural Rural Rural Rural Rural Rural Rural Rural Rural Contact with ticks Yes No Yes Yes Yes Yes Yes Yes Yes Milk* Yes Yes Yes Yes Yes Yes Yes Yes Yes ALT U/L 55 36 27 127 40 75 65 128 69 AST U/L 76 66 30 164 98 82 57 164 72 WBC 1 count (109/L) 2400 2300 2700 2000 1600 3300 4300 2000 5200 WBC 2 (109/L) 2100 1800 2000 2000 1600 3200 3500 2000 4500 Hb 12.2 11.6 13.9 14.9 13.8 14 11.2 14.9 13.6 Platelets 1 (109/L) 60 000 115000 139000 59000 70000 112000 112000 59000 120000 Platelets 2 (109/L) 42 000 61000 58000 55000 26000 90000 87000 59000 79000 CCHF PCR Positive Positive Positive Positive Positive Negative Negative Negative Negative Brucella STA 1/1280 1/640 1/1280 1/320 1/640 1/320 1/1280 1/640 1/1280 Blood Culture Sterile Sterile Sterile Sterile Sterile Sterile Sterile Sterile Brucella spp. *Consumption of unpasteurized milk and milk products ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, WBC 1: Initial white blood cell count on referral, WBC 2: Lowest white blood cell count, Hb: Haemoglobin, Platelet 1: Initial plate- let value, Platelet 2: lowest platelet value, STA: Standart tube agglutination. Normal values: AST: 15–37 IU/L, ALT: 30–65 IU/L, WBC: 4. 800–10.800mm3, Hb: 12–17g/dL, Platelet: 150000–400000mm3 Discussion This trial showed that zoonotic diseases can coexist due to similar modes of transmission and that brucellosis should not be ruled out in patients with CCHF. In this trial assessing the incidence of brucellosis in patients with CCHF, five patients had both of the diseases (4.16%). The PCR results of the four patients with a preliminary diagnosis of CCHF were negative, these four patients were diagnosed with brucellosis. We believe the patients with a preliminary diagnosis of CCHF should also be investigated for brucellosis. CCHF is a zo- onotic disease transmitted by Hyalomma ticks (1). Brucellosis is a zoonosis transmitted via body secretions of animals with brucellosis and by the consumption of milk and dairy products not boiled or pasteurized (11). Farmers, shep- herds, veterinarians, butchers, and laboratory staff are at risk for transmission (12). Both diseases are more commonly observed in people who deal with stockbreeding and live in rural areas. The most common patient com- plaints upon presentation with CCHF include fever, fatigue, diffuse body pain, and lack of appetite. Patients less commonly present with haemorrhage and rash compared to nausea and/ or vomiting, diarrhoea, and abdominal pain (1). Brucellosis is a disease that can involve any organ and tissue and therefore may man- ifest with different symptoms and findings (6, 12). Because the symptoms of brucellosis are nonspecific, differential diagnoses are dif- ficult and misdiagnoses are frequent. Patients most commonly present with fever, fatigue, sweating, joint pain, and lack of appetite. As the disease may involve various organs, the physical examination findings can also be var- J Arthropod-Borne Dis, December 2017, 11(4): 463–468 F Duygu et al.: Brucellosis in … 466 http://jad.tums.ac.ir Published Online: December 30, 2017 iable (6). The most common findings in bru- cellosis are fever, splenomegaly, hepatomegaly, lymphadenomegaly, and arthritis. This dis- ease may involve all the systems and may man- ifest with complications. Osteoarticular in- volvement is most common (13). In this trial, all patients developed at least two of the gen- eral infection signs of fever, fatigue, and lack of appetite. Leukopenia, thrombocytopenia, and anae- mia may develop in CCHF (14). Similar la- boratory findings can also be seen in brucel- losis as a result of bone marrow suppression (13). The negative CCHF PCR results and the positive Brucella STA test results obtained for four of the patients admitted with a pre- liminary diagnosis of CCHF with symptoms of fever, general infections signs, and bicy- topenia who presented due to tick contact demonstrated the importance of performing brucellosis tests in patients monitored for sus- pected CCHF. CCHF is seasonal and occurs between Apr and Sep, while brucellosis may occur in any season. Therefore, we believe that sea- sonal features should also be considered in establishing a diagnosis and that these two diseases could co-exist during the summer. The diagnosis of CCHF is based on virus iso- lation in the cell culture, serologic methods (immunofluorescence assays [IFAs], ELISAs) and reverse transcription PCR (1). The most common method of diagnosing brucellosis is the standard tube agglutination (STA) test. Standard tube agglutination test is an inexpen- sive, convenient method with a reported sen- sitivity of 94%. The gold standard in diagno- sis is the growth of bacteria in culture (15). All the patients were diagnosed based on PCR and/or IgM results. As the patients were hospitalized with a preliminary diagnosis of CCHF, the blood cultures could not be ob- tained routinely. One of the patients for whom blood culture was conducted after a brucel- losis diagnosis was established exhibited Brucella growth in the blood culture. Debate on the treatment of CCHF is on- going. While some publications show ribavirin is beneficial, others indicate its lack of effi- cacy (16-19). Ribavirin treatment has been shown not to reduce mortality in the treat- ment of CCHF (20). In this trial, ribavirin was not administered to patients diagnosed with CCHF; rather, symptomatic treatment was administered. Double or triple combina- tions of doxycycline, streptomycin, and ri- fampicin are recommended to treat brucello- sis (12). In this trial, all these three drugs were administered to the patients with oste- oarticular involvement. Other patients were given doxycycline and rifampicin. All pa- tients recovered. Brucellosis can show great similarity with hematologic and zoonotic dis- eases, such as CCHF (21-23). 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