viral replication once inside the skin, dengue virus binds to langerhans cells (a population of dendritic cells in the skin that identifies pathogens). the virus enters the cells through binding between viral proteins and membrane proteins on the langerhans cell, specifically, the c-type lectins called dc-sign, mannose receptor and clec5a. dc-sign, a non-specific receptor for foreign material on dendritic cells, seems to be the main point of entry. the dendritic cell moves to the nearest lymph node. meanwhile, the virus genome is translated in membrane-bound vesicles on the cell's endoplasmic reticulum, where the cell's protein synthesis apparatus produces new viral proteins that replicate the viral rna and begin to form viral particles. immature virus particles are transported to the golgi apparatus, the part of the cell where some of the proteins receive necessary sugar chains (glycoproteins). the now mature new viruses are released by exocytosis. they are then able to enter other white blood cells, such as monocytes and macrophages. the initial reaction of infected cells is to produce interferon, a cytokine that raises many defenses against viral infection through the innate immune system by augmenting the production of a large group of proteins mediated by the jak-stat pathway. some serotypes of the dengue virus appear to have mechanisms to slow down this process. interferon also activates the adaptive immune system, which leads to the generation of antibodies against the virus as well as t cells that directly attack any cell infected with the virus. various antibodies are generated; some bind closely to the viral proteins and target them for phagocytosis (ingestion by specialized cells and destruction), but some bind the virus less well and appear instead to deliver the virus into a part of the phagocytes where it is not destroyed but can replicate further. severe disease in antibody-dependent enhancement (ade), antibodies bind to both viral particles and fc gamma receptors expressed on immune cells, increasing the likelihood that the viruses will infect those cells. it is not entirely clear why secondary infection with a different strain of dengue virus places people at risk of dengue hemorrhagic fever and dengue shock syndrome. the most widely accepted hypothesis is that of antibody-dependent enhancement (ade). the exact mechanism behind ade is unclear. it may be caused by poor binding of non-neutralizing antibodies and delivery into the wrong compartment of white blood cells that have ingested the virus for destruction. there is a suspicion that ade is not the only mechanism underlying severe dengue-related complications, and various lines of research have implied a role for t cells and soluble factors such as cytokines and the complement system. severe disease is marked by the problems of capillary permeability (an allowance of fluid and protein normally contained within the blood to pass) and disordered blood clotting. these changes appear associated with a disordered state of the endothelial glycocalyx, which acts as a molecular filter of blood components. leaky capillaries (and the critical phase) are thought to be caused by an immune system response. other processes of interest include infected cells that become necrotic—which affect both coagulation and fibrinolysis (the opposing systems of blood clotting and clot degradation)—and low platelets in the blood, also a factor in normal clotting. diagnosis warning signs worsening abdominal pain ongoing vomiting liver enlargement mucosal bleeding high hematocrit with low platelets lethargy or restlessness serosal effusions the diagnosis of dengue is typically made clinically, on the basis of reported symptoms and physical examination; this applies especially in endemic areas. however, early disease can be difficult to differentiate from other viral infections. a probable diagnosis is based on the findings of fever plus two of the following: nausea and vomiting, rash, generalized pains, low white blood cell count, positive tourniquet test, or any warning sign (see table) in someone who lives in an endemic area. warning signs typically occur before the onset of severe dengue. the tourniquet test, which is particularly useful in settings where no laboratory investigations are readily available, involves the application of a blood pressure cuff at between the diastolic and systolic pressure for five minutes, followed by the counting of any petechial hemorrhages; a higher number makes a diagnosis of dengue more likely with the cut off being more than 1.5 to 3 per 1 cm2 (10 to 20 per inch2). the diagnosis should be considered in anyone who develops a fever within two weeks of being in the tropics or subtropics. it can be difficult to distinguish dengue fever and chikungunya, a similar viral infection that shares many symptoms and occurs in similar parts of the world to dengue. often, investigations are performed to exclude other conditions that cause similar symptoms, such as malaria, leptospirosis, viral hemorrhagic fever, typhoid fever, meningococcal disease, measles, and influenza. zika fever also has similar symptoms as dengue. the earliest change detectable on laboratory investigations is a low white blood cell count, which may then be followed by low platelets and metabolic acidosis. a moderately elevated level of aminotransferase (ast and alt) from the liver is commonly associated with low platelets and white blood cells. in severe disease, plasma leakage results in hemoconcentration (as indicated by a rising hematocrit) and hypoalbuminemia. pleural effusions or ascites can be detected by physical examination when large, but the demonstration of fluid on ultrasound may assist in the early identification of dengue shock syndrome. the use of ultrasound is limited by lack of availability in many settings. dengue shock syndrome is present if pulse pressure drops to ≤ 20 mm hg along with peripheral vascular collapse. peripheral vascular collapse is determined in children via delayed capillary refill, rapid heart rate, or cold extremities. while warning signs are an important aspect for early detection of potential serious disease, the evidence for any specific clinical or laboratory marker is weak. classification the world health organization's 2009 classification divides dengue fever into two groups: uncomplicated and severe. this replaces the 1997 who classification, which needed to be simplified as it had been found to be too restrictive, though the older classification is still widely used including by the world health organization's regional office for southeast asia as of 2011. severe dengue is defined as that associated with severe bleeding, severe organ dysfunction, or severe plasma leakage while all other cases are uncomplicated. the 1997 classification divided dengue into an undifferentiated fever, dengue fever, and dengue hemorrhagic fever. dengue hemorrhagic fever was subdivided further into grades i–iv. grade i is the presence only of easy bruising or a positive tourniquet test in someone with fever, grade ii is the presence of spontaneous bleeding into the skin and elsewhere, grade iii is the clinical evidence of shock, and grade iv is shock so severe that blood pressure and pulse cannot be detected. grades iii and iv are referred to as "dengue shock syndrome". laboratory tests graph of when laboratory tests for dengue fever become positive. day zero refers to the start of symptoms, 1st refers to in those with a primary infection, and 2nd refers to in those with a secondary infection. the diagnosis of dengue fever may be confirmed by microbiological laboratory testing. this can be done by virus isolation in cell cultures, nucleic acid detection by pcr, viral antigen detection (such as for ns1) or specific antibodies (serology). virus isolation and nucleic acid detection are more accurate than antigen detection, but these tests are not widely available due to their greater cost. detection of ns1 during the febrile phase of a primary infection may be greater than 90% sensitive however is only 60–80% in subsequent infections. all tests may be negative in the early stages of the disease. pcr and viral antigen detection are more accurate in the first seven days. in 2012 a pcr test was introduced that can run on equipment used to diagnose influenza; this is likely to improve access to pcr-based diagnosis. these laboratory tests are only of diagnostic value during the acute phase of the illness with the exception of serology. tests for dengue virus-specific antibodies, types igg and igm, can be useful in confirming a diagnosis in the later stages of the infection. both igg and igm are produced after 5–7 days. the highest levels (titres) of igm are detected following a primary infection, but igm is also produced in reinfection. igm becomes undetectable 30–90 days after a primary infection, but earlier following re-infections. igg, by contrast, remains detectable for over 60 years and, in the absence of symptoms, is a useful indicator of past infection. after a primary infection, igg reaches peak levels in the blood after 14–21 days. in subsequent re-infections, levels peak earlier and the titres are usually higher. both igg and igm provide protective immunity to the infecting serotype of the virus. in testing for igg and igm antibodies there may be cross-reactivity with other flaviviruses which may result in a false positive after recent infections or vaccinations with yellow fever virus or japanese encephalitis. the detection of igg alone is not considered diagnostic unless blood samples are collected 14 days apart and a greater than fourfold increase in levels of specific igg is detected. in a person with symptoms, the detection of igm is considered diagnostic. prevention a 1920s photograph of efforts to disperse standing water and thus decrease mosquito populations prevention depends on control of and protection from the bites of the mosquito that transmits it. the world health organization recommends an integrated vector control program consisting of five elements: advocacy, social mobilization and legislation to ensure that public health bodies and communities are strengthened; collaboration between the health and other sectors (public and private); an integrated approach to disease control to maximize the use of resources; evidence-based decision making to ensure any interventions are targeted appropriately; and capacity-building to ensure an adequate response to the local situation. the primary method of controlling a. aegypti is by eliminating its habitats. this is done by getting rid of open sources of water, or if this is not possible, by adding insecticides or biological control agents to these areas. generalized spraying with organophosphate or pyrethroid insecticides, while sometimes done, is not thought to be effective. reducing open collections of water through environmental modification is the preferred method of control, given the concerns of negative health effects from insecticides and greater logistical difficulties with control agents. people can prevent mosquito bites by wearing clothing that fully covers the skin, using mosquito netting while resting, and/or the application of insect repellent (deet being the most effective). while these measures can be an effective means of reducing an individual's risk of exposure, they do little in terms of mitigating the frequency of outbreaks, which appear to be on the rise in some areas, probably due to urbanization increasing the habitat of a. aegypti. the range of the disease also appears to be expanding possibly due to climate change. vaccine two types of dengue vaccine are available: qdenga and dengvaxia. on 5 december 2022 the european medicines agency approved qdenga, a live tetravalent attenuated vaccine for adults, adolescents and kids from four years of age, produced by takeda pharmaceutical company. in 2016 dengvaxia became commercially available in the philippines and indonesia. it has been approved for use by mexico, brazil, el salvador, costa rica, singapore, paraguay, much of europe, and the united states. dengvaxia is only recommended in individuals who have had a prior dengue infection or in populations where most (>80%) of people have been infected by age 9. in those who have not had a prior infection there is evidence it may worsen subsequent infections. for this reason prescrire does not see it as suitable for wide scale immunization, even in areas where the disease is common. dengvaxia is produced by sanofi. it is based on a weakened combination of the yellow fever virus and each of the four dengue serotypes. studies of the vaccine found it was 66% effective and prevented more than 80 to 90% of severe cases. this is less than wished for by some. in indonesia it costs about us$207 for the recommended three doses. given the limitations of the current vaccine, research on vaccines continues, and the fifth serotype may be factored in. one of the concerns is that a vaccine could increase the risk of severe disease through antibody-dependent enhancement (ade). the