Despite improvements in health, epidemics of infectious diseases continue to occur, and new diseases emerge and old diseases reemerge (113). Mosquito-borne flavivirus diseases are currently considered reemerging infections because of the increase in the incidences of yellow fever and, mainly, dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) observed in the last few years (30, 86). The dengue syndrome is an acute febrile viral exanthem, accompanied by headache, myalgia, anorexia, gastrointestinal disturbances, and postration, caused by viruses transmitted by mosquitoes (43). DHF is a severe febrile disease characterized by abnormalities of hemostasis and increased vascular permeability. DSS is the result of a hypovolemic shock observed in some DHF cases. DHF/DSS represents the severe form of dengue fever (52). The disease is caused by any one of the four distinct serotypes (1 to 4) of the dengue virus (52, 114). These viruses are members of the family Flaviviridae; they have a common morphology and genomic structure, and all members share common antigenic determinants. The four dengue virus serotypes are classified as a complex on the basis of clinical, biological, and immunological criteria. Dengue virus complex-specific antigenic determinants have been demonstrated by using neutralization assays, which also can differentiate the dengue virus complex into four antigenically distinct dengue virus serotypes, since each serotype presents a type-specific determinant (49, 52). The flaviviruses are transmitted by mosquitoes of the Stegomia family, mainly Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans (52, 99). This is a highly urbanized mosquito, breeding in water stored for domestic use or collected rainwater. A jungle cycle has been proposed to exist in Southeast Asia, since there is a high rate of dengue transmission among different species of monkeys (52, 105). The genomic RNA of dengue viruses is single stranded and approximately 11 kb in length. The RNA is infectious and, as in the rest of the flaviviruses, it has a single open reading frame (103). The order of proteins encoded in the long open reading frame is 59-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5-39. The mature virion contains three structural proteins: C, the nucleocapside or core protein of 13.5 kDa; M, a membrane-associated protein of 8 kDa; and the E (envelope) protein of 51 kDa. The E protein has the sites for viral attachment to and transport through host cell plasma membranes. Functional domains responsible for the neutralization and hemagglutination of goose erythrocytes are associated with the E protein. It contains epitopes specific for serotype, dengue complex, and group (6, 48, 103, 115). Considering the technology currently used for the diagnosis of dengue viruses, a case definition in which laboratory confirmation is emphasized has been proposed. The laboratory criteria for confirmation of the infection and the disease include the isolation of dengue virus from serum and/or autopsy samples, the demonstration of a fourfold or greater increase in the titer of immunoglobulin G (IgG) or IgM antibody to one or more dengue virus antigens in paired serum samples, or the demonstration of dengue virus antigen in autopsy tissue or serum samples by immunohistochemistry, by immunofluorescence, or by the detection of the viral nucleic acid (98).

https://cvi.asm.org/content/cdli/3/6/621.full.pdf

Advances in dengue diagnosis.

Four primer pairs were selected on the basis of the published sequence data of four dengue virus serotypes so that each unique target sequence size could be amplified for each serotype by polymerase chain reaction. The procedure consists of (i) RNA preparation, (ii) reverse transcription, and (iii) polymerase chain reaction, all of which could be completed within 2 h in a single tube for each specimen. The amplified sequence size revealed by ethidium bromide-stained agarose gel electrophoresis was unique for each serotype, using infected culture fluid of isolates from dengue fever or dengue hemorrhagic fever patients in Thailand, Indonesia, and the Philippines as well as from prototype viruses, thus facilitating simultaneous identification and typing.

/pdf/rapid-identification-of-dengue-virus-serotypes-by-using-5bqhg2nwco.pdf

Rapid identification of dengue virus serotypes by using polymerase chain reaction.

Dengue is the most important disease caused by an arbovirus (1, 2, 3 and 4 serotypes) worldwide, especially in the tropical and sub-tropical regions. Its clinical manifestations range from asymptomatic infections to a severe disease characterized by hemorrhage and shock. The incidence of dengue virus activity in the Americas has substantially increased from 1980 to 1994. In Brazil, the increase in the incidence of dengue is especially linked to the dissemination of Aedes aegypti. Thus, a rapid and accurate dengue diagnosis is of paramount importance for effective control of dengue outbreaks [8]. Five serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. The limitations of these techniques are the high cross-reactivity observed with these tests. Four methods of viral isolation have been routinely used for dengue viruses: intracerebral inoculation of newborn mice, inoculation on mammalian cell cultures, intrathoracic inoculation of adult mosquitoes, and inoculation on mosquito cell cultures. In recent years, several new diagnostic techniques have been developed and have proven very useful in dengue diagnosis, such as: nucleic and acid hybridization, RT-PCR. Currently, dengue diagnosis is based on serology, viral isolation and RNA detection. Enzyme-linked immunosorbent assays (ELISA) are still the most widely used technique for serological diagnosis, but they do not identify the dengue virus serotype responsible for the current infection, so molecular techniques may soon assume a very important role in dengue diagnosis. RT-PCR is definitely the most satisfactory test that can be used on these infections, since it has been shown to be able to detect dengue viruses up to the 10th day after the onset of the symptoms.

http://www.scielo.br/pdf/bjid/v8n6/a02v08n6.pdf

Dengue: a review of the laboratory tests a clinician must know to achieve a correct diagnosis

Serodiagnosis of flaviviral infections and vaccinations in humans.

Serum specimens from patients with imported dengue fever and dengue hemorrhagic fever were directly subjected to reverse transcription and polymerase chain reaction (RT‐PCR) without any RNA purification. The amplified virus genome was detected within 3 hours. The results of PCR corresponded closely to the results of virus isolation using cultured mosquito cells, suggesting that direct RT‐PCR procedure greatly facilitates rapid diagnosis of dengue infection. © 1994 Wiley‐Liss, Inc.

Rapid detection of virus genome from imported dengue fever and dengue hemorrhagic fever patients by derect polymerase chain reaction

sera fromsecondary dengue infections. Additionally, 32of76single serafrompatients were positive fordengue IgM;these serawerepreviously uninterpretable bythehemagglutination inhibition test, asonly asingle serum specimen was available. No false-positive results were obtained withsera thatwere negative bythe hemagglutination inhibition testfordengue virus. Crudedengue virushemagglutinin preparations couldbe usedwithout purification. Dengue-specific IgGdidnotinterfere withtheresults, nor was thereany cross-reactivity between dengue hemagglutinins andIgMspecific forother viruses. Somecross-reactivity ofthe dengue-specific IgMwas observed withJapanese encephalitis virus hemagglutinins, butthis didnotpresent any problems intheinterpretation ofresults. Thistest isspecific, inexpensive, highly reproducible, andsimple to perform. Denguefever (DF)anddengue hemorrhagic fever (DHF) contribute significantly tomorbidity andmortality inmany parts ofthetropical world. An important factor inthe management ofDF andDHF andintheprevention and control ofoutbreaks istheavailability ofarapid, sensitive, specific, andeconomical laboratory diagnostic test toconfirmdengue infection. However, currently available tests based onvirus isolation andserology arestill unsatisfactory inthis regard. Withregard toserology, mostlaboratories use conventional methods suchashemagglutination inhibition (HAI), thecomplement fixation test, andtheneutralization test todiagnose dengue virus infections. Theseserological tests areofdiagnostic importance ifaseroconversion or fourfold orgreater rise inantibody titers between acute- and convalescent-phase seracanbedemonstrated. Veryoften thecollection ofpaired seraisnotwellspaced toshowarise intiter ofdiagnostic value, oronlyasingle serumspecimen iscollected. Itwouldthusbeofvalue todevelop aspecific andeconomical testwhichwouldbepositive early inthe infection and,preferably, require onlyasingle specimen. Thedetection ofdengue-specific immunoglobulin M (IgM) maybeconsistent withthese considerations. Because IgMgenerally appears early intheonsetof disease andisrelatively transient, its detection wouldbean indication ofanongoing orrecent infection. Conventional methods ofdetecting IgMantibody require thephysical separation ofIgMfromIgG.Thesemethods, besides being costly, aretedious toperform andtime-consuming. A significant advance intherapid diagnosis ofviral infections was thedevelopment ofsolid-phase immunoassays forthedetection ofvirus-specific IgMantibody (4,6,15,16). Thebasis

T. Pang

Papers

Hemadsorption Immunosorbent Technique fortheDetection of DengueImmunoglobulin M Antibody