Ebola viruses may cause severe hemorrhagic fever outbreaks in humans with a death rate up to 90%. The two initial outbreaks occurred in 1976 in Sudan and in the Democratic Republic of the Congo (near to the Ebola River) [2International Study Team. Ebola haemorrhagic fever in Sudan, 1976 Bull World Health Organ 1978; 56: 247-70.
[PMID: 307455] , 3Ebola haemorrhagic fever in Zaire, 1976 Bull World Health Organ 1978; 56(2): 271-93.
[PMID: 307456] ].

There are five different species of the Ebola virus named Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, Taï Forest ebolavirus (formerly Côte d’Ivoire ebolavirus) and Reston ebolavirus [4Formenty P, Hatz C, Le Guenno B, Stoll A, Rogenmoser P, Widmer A. Human infection due to Ebola virus, subtype Côte d’Ivoire: clinical and biologic presentation J Infect Dis 1999; 179(Suppl. 1): S48-53.
[http://dx.doi.org/10.1086/514285] [PMID: 9988164] , 5Miranda ME, Ksiazek TG, Retuya TJ, et al. Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996 J Infect Dis 1999; 179(Suppl. 1): S115-9.
[http://dx.doi.org/10.1086/514314] [PMID: 9988174] ]. The three first viruses are associated with several Ebola disease (EVD) outbreaks in Africa.

Although, the wildlife reservoir has not been definitively found, the possible natural hosts for Ebola virus are some species of bats.

EVD is introduced into the human population through direct contact with infected animal body fluids or vector bites. In non-endemic countries, EVD spreads through direct contact with the body fluids of infected patients. After the incubation period (2 to 21 days), EVD appears with fever and malaise, and other non-specific symptoms and signs (such as myalgia, weakness, headache, muscle pain, sore throat, diarrhea and vomiting). Rash and hemorrhagic symptoms are visible in 30-50% of patients with EVD, though multi-organ dysfunction leading to shock and death may be present in serious forms.

The Zaire Ebola virus has caused most outbreaks in Central Africa since their apparition in 1976. The 2014 Ebola outbreak is the first in West Africa. Until November 2 2015, 15246 cases have been confirmed in the laboratory and 11314 patients have died.

Marburg virus, which causes Marburg hemorrhagic fever (MHF), and Ebola virus are the two members of the Filoviridae family that can cause outbreaks with high fatality rates [6Gear JS, Cassel GA, Gear AJ, et al. Outbreake of Marburg virus disease in Johannesburg BMJ 1975; 4(5995): 489-93.
[http://dx.doi.org/10.1136/bmj.4.5995.489] [PMID: 811315] ]. This disease was first identified in 1967 after the outbreaks in Marburg, Frankfurt and Belgrade [7Martini GA. Marburg agent disease: in man Trans R Soc Trop Med Hyg 1969; 63(3): 295-302.
[http://dx.doi.org/10.1016/0035-9203(69)90001-7] [PMID: 5815873] ]. Travelers are at low risk of infection, but occasionally these viruses can be imported into non-endemic countries.

In Africa, some species of bats are considered natural hosts for Marburg virus. During the first Marburg outbreak, the etiology of human infection was traced to the laboratory handling of African green monkeys imported from Uganda. Since then, some outbreaks and sporadic cases have been reported in several countries such as Angola, Democratic Republic of the Congo, Kenya, South Africa and Uganda [7Martini GA. Marburg agent disease: in man Trans R Soc Trop Med Hyg 1969; 63(3): 295-302.
[http://dx.doi.org/10.1016/0035-9203(69)90001-7] [PMID: 5815873] ]. In 2008, two cases of MHF were reported in travelers; the infection was acquired as a result of exposure to caves inhabited by bats in Uganda. Transmission is mainly caused through direct contact with body fluids of infected persons. Also, transmission by infected semen can occur as well as transmission to health care workers while administering treatments to infected patients.

After the incubation period (which ranges from 2 to 21 days), MHF begins suddenly with fever, headache and malaise, myalgia, arthralgia, abdominal pain, diarrhea, nausea and vomiting, conjunctiva injection and a relative bradycardia. Most of the patients develop severe hemorrhagic features in multiple body parts between 5-7 days. Around the fifth day, a maculo-papular rash could also be seen. Involvement of the CNS can result in irritability and confusion.

Yellow fever (YF) is a viral hemorrhagic disease caused by a flavivirus and transmitted by infected mosquitoes [8Teichmann D, Grobusch MP, Wesselmann H, et al. A haemorrhagic fever from the Côte d’Ivoire Lancet 1999; 354(9190): 1608.
[http://dx.doi.org/10.1016/S0140-6736(99)09233-8] [PMID: 10560677] ]. The term “yellow” refers to the jaundice that can be seen in some persons, and up to 50% of the infected patients could die without due treatments. This infection is endemic in Africa and South America; outbreaks have been identified more frequently in Western Africa. In the jungle cycle, the main hosts of this virus are monkeys, whereas in the urban cycle humans are the reservoirs. Aedes mosquitos’ species can serve as vectors of YF in Africa, and the species involved depends on the geographical location and type of transmission cycle. Nine hundred million persons from 44 endemic countries in Africa and Latin America are at risk of acquiring this infection. Each year, an estimated 200,000 new cases of YF worldwide are diagnosed causing 30,000 deaths. Small numbers of imported cases occur every year in non-endemic countries. In December 2010, 190 cases and 48 deaths were reported in Northern Uganda, affecting mainly males aged 20 to 34 years.

YF should be suspected in persons without vaccination who have recently traveled to endemic areas [9Schwartz F, Drach F, Guroy ME. From the Centers for Disease Control and Prevention. Fatal yellow fever in a traveler returning from Venezuela, 1999 JAMA 2000; 283(17): 2230-2.
[http://dx.doi.org/10.1001/jama.283.17.2230-JWR0503-2-1] [PMID: 10807371] ]. International regulations require vaccination against YF, but some non-vaccinated travelers in endemic areas are able to evade controls.

After the incubation period (3 to 6 days), the infection can develop in two phases. The first phase is an acute illness presented with fever, muscular pain and loss of appetite, nausea, vomiting, shivers, backache, and headache. Most patients improve at this stage; the symptoms disappear after 3-4 days. The second phase is developed in 15% of the infected persons with symptomatology of kidney failure such as jaundice, abdominal pain, and vomiting. Also, bleeding can be present in several locations; 50% of the patients may die within 10-14 days.

Dengue is a viral infection caused by viruses of the Flaviviridae family and endemic in tropical and sub-tropical areas. Dengue was first found in 1950 in the Philippines and Thailand. Four different serotypes of the virus (namely, DEN-1, DEN-2, DEN-3 and DEN-4) have been identified. Whenever dengue infection is developed, lifelong immunity against a specific serotype is produced, though cross-immunity to the other serotypes may be demonstrated (this is temporary and partial). Severe dengue is produced when further infections by other serotypes occur.

Dengue disease is transmitted through bites of Aedes mosquitoes (e.g.,Aedes aegypti, Aedes albopictus); it is widely spread in Africa, America, Asia, the Pacific and the Caribbean regions. Last years, the incidence of dengue increased quickly worldwide; the transmission has grown above all in urban and semi-urban areas. Currently, dengue is a major international public health problem because over 40% of the world’s population is at risk of infection. This disease is endemic in more than 100 countries; the WHO estimates that 50-100 million of dengue infections occur every year worldwide. Data from 2010 reported 1.6 million cases of dengue in American countries (49,000 of them are severe). In 2010, in Europe, 1622 cases of dengue were reported, particularly in Germany, Sweden, France, and Belgium, and local transmission was reported in France and Croatia [10Gjenero-Margan I, Aleraj B, Krajcar D, et al. Autochthonous dengue fever in Croatia, August-September 2010 Euro Surveill 2011; 16(9): 19805.
[PMID: 21392489] , 11Domingo C, Niedrig M, Gascón J, et al. Molecular surveillance of circulating dengue genotypes through European travelers J Travel Med 2011; 18(3): 183-90.
[http://dx.doi.org/10.1111/j.1708-8305.2011.00501.x] [PMID: 21539658] ]. In 2012, an outbreak of dengue occurred in Madeira (Portugal) resulting in approximately 2000 cases with imported cases being detected in 10 other European countries. In 2013, some cases occurred in Florida (U.S.) and the Yunnan province of China. Each year, an estimated 500,000 people with severe dengue require hospitalization and about 2.5% of those affected persons die.

The incubation period is about 4-10 days; the disease starts with high fever (40°C), severe headache, eyes paint, muscle and joint paints, nausea, vomiting, swollen glands and rash. The prognosis of severe dengue can be fatal due to severe bleeding, respiratory distress, fluid accumulation, plasma leaking, or organ failure. Severe symptoms can include severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, gastrointestinal bleeding, restlessness and extreme fatigue.

Lassa fever (LF) is a disease caused by a member of the Arenavirus family of viruses called Lassa virus. This virus is transmitted to humans through direct contact with food or objects contaminated with rodent excreta, though human-to-human infections and laboratory transmission could also occur, primarily through direct contact with body fluids.

LF is an endemic disease in West Africa, highly prevalent in Nigeria, Sierra Leone, and Liberia. The name “Lassa” came to existence in Nigeria, where LF was first recognized, in 1969 [12Buckley SM, Casals J. Lassa fever, a new virus disease of man from West Africa. 3. Isolation and characterization of the virus Am J Trop Med Hyg 1970; 19(4): 680-91.
[PMID: 4987547] ]. The majority of persons at risk are those living in rural areas of the West African countries [13McCormick JB. Epidemiology and control of Lassa fever Curr Top Microbiol Immunol 1987; 134: 69-78.
[http://dx.doi.org/10.1007/978-3-642-71726-0_3] [PMID: 3581899] ]; though sporadic cases have been reported in the Netherlands, the U.S., Japan and Canada [14Lassa fever, imported case, Netherlands Wkly Epidemiol Rec 2000; 75(33): 265-72.
[PMID: 10974948] , 15Amorosa V, MacNeil A, McConnell R, et al. Imported Lassa fever, Pennsylvania, USA, 2010 Emerg Infect Dis 2010; 16(10): 1598-600.
[http://dx.doi.org/10.3201/eid1610.100774] [PMID: 20875288] ].

The majority of patients are asymptomatic, but 20% may develop serious multi-system disease, and up to 15% of them could die as a consequence of this disease. The main symptoms and signs of LF are fever, headache, sore throat, myalgia, dysphagia, dry cough, chest pain and abdominal pain. Complications can occur with severe symptoms such as face and neck edema, pleural and pericardial effusion, respiratory distress, encephalopathy and hemorrhage in several locations. LF is more severe during pregnancy.

Crimean-Congo hemorrhagic fever (CCHF) is a disease caused by Nairovirus, which belongs to the Bunyaviridae family. This disease was first noticed in Russia as Crimean hemorrhagic fever in 1945 and in the Democratic Republic of the Congo as Congo virus infection in 1956. The virus causes serious hemorrhagic fever outbreaks with a case fatality rate that ranges from 10 to 40%.

CCHF is endemic in Africa, the Balkans, the Middle East and Asian countries, and can occur in large areas of Eastern Europe, the Mediterranean region, Western Asia and Africa [16Gear JH, Thompson PD, Hopp M, et al. Congo-Crimean hemorrhagic fever in South Africa S Afr Med J 1982; 62: 576-80.
[PMID: 7123427] , 17Swanepoel R, Shepherd AJ, Leman PA, et al. Epidemiologic and clinical features of Crimean-Congo hemorrhagic fever in southern Africa Am J Trop Med Hyg 1987; 36(1): 120-32.
[PMID: 3101525] ]. In Europe, some cases of human infections have been found in Albania, Armenia, Bulgaria, Serbia, Turkey and other countries. Two confirmed cases of CCHF were reported in 2010 in Bulgaria. In 2010, the CCHF viral genome was identified in Hyalomma ticks collected in Cáceres (Spain), which suggests that the virus might produce disease in other regions than those considered as endemic in Europe [18Estrada-Peña A, Palomar AM, Santibáñez P, et al. Crimean-Congo hemorrhagic fever virus in ticks, Southwestern Europe, 2010 Emerg Infect Dis 2012; 18(1): 179-80.
[http://dx.doi.org/10.3201/eid1801.111040] [PMID: 22261502] ].

The hosts of this virus include both wild and domestic animals such as sheep, goats, and cattle. Animals can be infected by the bites of infected ticks, above all those of the genus Hyalomma. The main modes of transmission from animals to humans include direct contact with blood or tissues of infected animals, as well as inoculation of the virus through the bites of infected ticks. Person-to-person transmission may also occur from direct contact with body fluids of infected patients.

The incubation period ranges from 2 to 9 days; the main symptoms and signs are fever, myalgia (backache, headache, and muscle pain), dizziness, sore eyes, and photophobia. Other symptoms include nausea, vomiting, diarrhea, and abdominal pain. There may also be meningeal signs with confusion and aggression, and after 2-4 days of sleepiness, depression and lassitude may appear.

Other clinical signs include tachycardia, hepatomegaly, and lymphadenopathy. Petechyal rash, ecchymoses, epistaxis or bleeding in other locations could also be present. The mortality rate is approximately 30%.

Rift Valley fever is an acute viral zoonosis that affects mainly sheep, goats and cattle and may also affect humans. RVF virus is a member of the Phlebovirus genus (family Bunyaviridae). In 1930, this virus was first identified in Kenya. In 1997-1998, the last big RVF outbreak was reported in Kenya, Somalia and Tanzania [19An outbreak of Rift Valley Fever, eastern Africa, 1997-1998 Wkly Epidemiol Rec 1998; 73(15): 105-9.
[PMID: 9577708] ]. In 2000, some cases of RVF were also reported in Saudi Arabia and Yemen [20Outbreak news: Riff Valley fever, Yemen (update) Wkly Epidemiol Rec 2000; 75: 321.
[PMID: 11050897] ]. However, an RVF outbreak in South Africa, which started in 2008-2009 and continued during the first half of 2010, resulted in a total of 236 human cases [21Rift Valley fever, South Africa Wkly Epidemiol Rec 2010; 85(20): 177-8.
[PMID: 20480979] ]. Mauritania also reported human cases in 2010.

RVF may be an occupational disease acquired through direct contact with infected animals and their products. RVF may also be transmitted to humans by Aedes bites and by ingesting unpasteurized and uncooked milk from infected animals. No person-to-person transmission has been documented to date and there is no evidence of outbreaks of RVF in urban areas.

This disease is usually found in Eastern and Southern Africa, but also in most countries of sub-Saharan Africa, Madagascar, Saudi Arabia and Yemen.

The incubation period for RVF ranges from 2 to 6 days and asymptomatic or mild infections are common. In symptomatic cases, the disease is characterized by fever, muscle and joint pain, and headache. Some patients also develop photophobia, retro-orbital pain, loss of appetite, nausea and vomiting, and maculopapular rash. However, less than 5% of the patients can develop a severe form of the disease characterized by three different syndromes involving meningo-encephalitis (< 1%), ocular disease (0.5-2%) or hemorrhagic fever (< 1%).

The main responsibility of a Laboratory director is to determine and assess the risk. He/she must meet all requirements and establish control measures to ensure safe practices and procedures.

Potentially infectious samples (e.g., blood, cerebrospinal fluid, pleural and peritoneal fluid) should be routinely handled and tested safely in the laboratory. The majority of hemorrhagic fever viruses are considered as biosafety level four (BSL-4) pathogens, except YF and Dengue. All laboratory workers and other healthcare personnel collecting or handling these samples must wear appropriate personal protective equipment (PPE) and follow all safety recommendations for handling samples. Specimen collection should be carried out by trained personnel following strict protocols adapted for each hospital or healthcare institution. These workers should wear gloves, gowns that are impermeable, eye protection (e.g., full face shield), and a mask to cover all of the nose and mouth area. Shoes and leg covers could be appropriate in certain circumstances such as if specimen collection is performed in a highly contaminated room.

The essential samples for virus isolation are a specimen of venous blood, a sample of urine, and a throat specimen and should be limited to the minimum amount necessary. Venous blood samples should be collected with care avoiding needle sticks. Safe procedures should be followed: the blood should be placed in an appropriate and safe container, and needles should be immediately discarded. Urine samples should be placed in an appropriate container with buffers (e.g., bovine serum albumin at 10% or human serum albumin at 1%). Finally, throat swabs should be placed in adequate containers in the same buffers as mentioned above. These specimens should be placed in double sealed plastic bags; these containers should be disinfected outside.

Clinical specimens should be placed and processed in a class II biological safety cabinet (BSC) following universal biosafety level 3 practices. It is also a good practice to pretreat serum samples with heat inactivation at 56ºC and polyethylene glycol p-tert-octylphenyl ether (Triton ^® X-100) [29Bhagat CI, Lewer M, Prins A, Beilby JP. Effects of heating plasma at 56 degrees C for 30 min and at 60 degrees C for 60 min on routine biochemistry analytes Ann Clin Biochem 2000; 37(Pt 6): 802-4.
[http://dx.doi.org/10.1258/0004563001899997] [PMID: 11085629] ]. Blood smears should be fixed in solvents in order to eliminate the infectivity; blood cultures should be preferably prepared in a closed system. It is essential to avoid aerosol production or splashing.

Routine cleaning and disinfecting procedures should be used for automated analyzers. These should be disinfected with 500 parts per million solutions of sodium hypochlorite. However, a new approach in specimens handling in the laboratories may be the use of point-of-care (POC) devices in the private room of the patients for critical analysis. This procedure could avoid the possible contamination of automated analyzers and other devices, surfaces and other sites of the laboratory. If POC instruments are to be used, the clinical laboratory must ensure that these devices can be used in critical care units. The laboratory should establish the accuracy, sensitivity, specificity, range of test results and reference intervals, as well as a quality control program for these instruments.

In order to send specimens to reference centers, the samples should be placed for transport in sealed containers. These should be resistant to low temperatures (-80º C). This primary container should be surrounded by absorbent material allowing absorption of the content in case the container breaks, and then should be placed in a secondary impermeable metal container with tape. Relevant information on the sample should be labeled on the outside of the secondary container. The secondary container should be placed in a definitive secure box.

The sample should be transported on dry ice via a carrier that should be compliant to regulations for transporting biological specimens.

VHF viruses are susceptible to a broad range of hospital disinfectants. Surfaces and objects contaminated with body fluids should be disinfected using standard procedures. Common hospital disinfectants or a 1:100 dilution of sodium hypochlorite could be used for disinfection. For surfaces, a 1:10 dilution of sodium hypochlorite should be used. Feces, vomitus and other medical fluids can be thrown away in the sanitary. Solid medical devices such as contaminated syringes and needles should be discarded in rigid containers and then incinerated.