3DisclaimerThe points made in this presentation are solely the views/opinions of AL and do not reflect the views/opinions of the US Government, US Department of Defense, US Army or the US Army Medical Research and Materiel Command .
4Take Home Points Mosquito borne illness Not spread person-to-personFirst infection can be a real bad experienceSecond infection can be deadlyNo antiviral treatmentNo vaccine (yet)
5Case (1)25 y/o male Indiana Jones type presents to your clinic in Ft. Bragg NC with c/o headache, abdominal pain, nausea and vomiting for the past 24 hours. Took pepto bismol and tylenol without relief.In the last 6 months he has been OCONUS (subsaharan Africa , Latin/South America and SE Asia) lived austerely (avoided hotels), swam in the ocean, ate adventurously, suffered numerous different insect bites, partook of some “horizontal refreshment” with local talent, and volunteered to be a cow herder for 2 weeks in the Pampas. He has 2 cats, a dog, tropical fish and several ferrets as pets at home. 3 weeks ago he cleaned out his aquarium, and stated it was a “bloody chore”. He did not take appropriate prophylaxis prior to/during the trip.What do you do?A) Bellyache, GOMER, dischargeB) Give him extra-strength PB and discharge with instruction to f/u with primary HCP next week,C) Admit, evaluate for, among other things, malaria, dengue and RMSFD) Consult psychiatry
6Introduction to Dengue Definition: Mosquito-borne flaviviral disease.Etiology:Infection with one of four types of dengue virus:DEN-1DEN-2DEN-3DEN-4Transmission:Vector: Aedes mosquitoAedes aegyptiAedes albopictusBlood TransfusionOrgan transplantationNo person to person transmission documentedAn electron image of mature Dengue-2 virus particles replicating in five-day-old tissue culture cells. The original magnification is 123,000 times. This is the Aedes aegypti vector taking a blood meal. It can be distinguished by the white bands on the thorax and legs. Also an efficient vector for YF and chikungunya, it typically breeds near human habitation, using relatively fresh water from sources like water jars, vases, discarded containers, cocunut husks and old tires. It usually inhabits dwellings and bites during the day.
7Dengue Virus Flavivirus (single-stranded RNA virus) Spherical, nm (dia.) viral particle3 Structural (E, C, M) proteins7 Nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5)4 serotypesDENV 1 through 4Multiple genotypes per serotypeCommon progenitor 1,000 years agoSerotypes have further divergence62 to 67% homology based on amino acid sequenceVarying pathogenicity based on serotypeDengue is a flavivirus which is similar phylogenetically to WNV and yellow Fever.The arose from a commom progenitor 1000 years ago.Divergence started about 125 to 300 years ago. DEN 4 is the most divergentThe four dengue viruses could have been treated as different viruses.DENV 4 appears to be most mild. DENV2 and 4 appear to be asociated with more sever disease in secondary infection. DENV1 and 3 cause more severe disease in primary infection
8History of DengueClinical descriptions date as far back as 992 AD in ChinaDavid Bylon (Batvia) in 1779“knokkelkoorts” --- joint feverBenjamin RushTermed “breakbone fever”Comes from Swahili “ka dinga pepo”meaning a sudden cramp like seizureand plagueFirst epidemics were first well codumented in 1779 to Likely original descriptions may have been referring to chikungunya.Viral etiology suggested in 1900’s by Ashburn and Craig making it the second viral human pathogen identified (Yellow fever by Walter Reed)Thomas S Advances in Virus Research 2003.Kyle J Annu Rev Microbiol :71-92.
9History of Dengue (2)Viral etiology suggested in early 1900’s by Ashburn and CraigVirus types 1 and 2 isolated during World War II1956 outbreak in Manila led to identification of Den-3 and DEN-4Dengue hemorrhagic fever recognized since 1950’sFirst epidemics were first well codumented in 1779 to 1780.Viral etiology suggested in 1900’s by Ashburn and Craig making it the second viral human pathogen identified (Yellow fever by Walter Reed)AL: Could point out that Ashburn and Craig were Army Docs; other Army (WRAIR) docs involved in early (and recent) studies include Siler (established period of infectivity for humans, mosquitoes, incubation period), Sabin (discovered dengue hemagglutinins, distinguished 2 dengue virus serotypes, demonstrated homotypic protective immunity, discovered dengue virus relation to other flaviviruses), Halstead (advanced theory of antibody-dependent immune enhancement, demonstrated blood monocytes as dengue targets, Rhesus monkey work), Bancroft (one of the first investigators who worked on DEN-2 vaccines), Russell (developed dengue PRNT, described the pathogenesis and antibody response of DF/DHF/DSS), Innis (vaccine studies), Vaughn (vaccine studies).Thomas S Advances in Virus Research 2003.Kyle J Annu Rev Microbiol :71-92.
11Vector Aedes aegypti and Ae. albopticus Highly susceptible to dengueEfficient vectorsPrefer human bloodDaytime feeder: interrupted, between egg laying;Bite goes unnoticedMultiple bites per blood meal; one mosquito can infect several personsAdapted to urban life; breeds in freshwater containersEND RESULT: RAPID TRANSMISSION, EXPLOSIVE EPIDEMICSBelieved that Aedes originated in jungles of Africa. Same vector as yellow fever and chikungunyaAlbopticus is Asian tiger mosquitoUses relatively fresh water from sources like water jars, vases, discarded containers, cocunut husks and old tires. It usually inhabits dwellings and bites during the day.AL: a frequent question I’m asked is, can other mosquitoes (eg, Anopheline sp.) cause dengue? These others can harbor the mosquito, but no significant human transmission has occurred.
12LarvaeA water sample is teeming with mosquito larvae after it was collected from a fountain outside a vacant house July 15, 2010 in Miami Beach, Fla. Miami-Dade County health officials are reporting the first suspected local case of dengue fever, a potentially serious mosquito-borne illness that had once disappeared from the United States
14Clean, Still Stagnant water Breeding sitesClean, Still Stagnant water
15World distribution of dengue viruses and their mosquito vector, Aedes aegypti, in 2005 Dengue occurs in tropical and subtropical areas in the world. The yellow areas are those at risk for epidemic dengue--that is, those areas infested with Aedes aegypti or other mosquito vectors of dengue. The red areas are those countries with recent dengue activity.The tropical zone of the world between 350N and 350S latitude and area not over 1,000 ft. above sea level is the usual habitat, the areas are marked by monsoon-rains.
16Epidemiology Assessment Leading arboviral (mosquito-borne) infectionMajor health problem in the subtropics and tropics (~35oN and ~35o S)Transmission (endemic) in ~ 100 countriesSoutheast Asia, India, Middle East, Caribbean, Central and South America, Australia, South and Central Pacific, AfricaRecent suspected dengue outbreaks in Yemen, Pakistan, Saudi Arabia, Madagascar, Sudan, Cape Verdebillion people at risk for infection (up to 40% of the world’s population)million infections annually~500,000 cases of DHF annuallyUp to 25,000 deaths annuallyMostly childrenSource:
17DENGUE is present between 35 degrees north and south of the equator
18Total Dengue Cases and Deaths, 2003-2008 Source: DengueNet
19Economic Assessment Significant Economic Burden Direct and Indirect costsSE Asia: 1,300 disability-adjusted life yearsDHF in Thailand: $19 to $51 (US) Million/yearSimilar to TB, other childhood and tropical diseases1981 Cuban DF/DHF/DSS epidemic$103 Million (US), including control measures ($43 Million) and medical services ($41 Million)Puerto Rico DF/DHF/DSS annual$150-$200 Million (US)
212012 Africa and the Indian Ocean Islands Atlantic Islands Mogadishu, Somalia (August 2012), eastern Kenya (May 2012), and Mandera, Kenya. The Kenyan Ministry of Health and local health officials are working with local hospitals and clinics to monitor the situation.Atlantic IslandsAccording to the European Centre for Disease Prevention and Control (ECDC), 1,148 cases of dengue (517 cases laboratory confirmed) have been reported as of November 4, 2012, on the Portuguese island of Madeira. Fifty-seven people have been hospitalized. No deaths have been reported.South Pacific and Southeast AsiaFederated States of Micronesia (September 2011 to April 2012), 1,200 cases and two deaths. Yap Main Island (July 2012), and Yap Outer Islands.Confirmed dengue cases have been reported in US travelers returning from destinations in Asia, specifically the Philippines and Thailand. Singapore, Malaysia, Cambodia, Taiwan, the Philippines, Vietnam, India, Sri Lanka, and Thailand are among the countries reporting dengue activity in 2012.Australia also continues to report sporadic dengue activity in areas of northern Queensland. For more information about dengue reports, visit the World Health Organization (WHO) Western Pacific Regional Office and the WHO South-East Asia Regional Office websites.The Americas and the CaribbeanIn 2012, dengue cases have been reported in most countries in Latin America. Confirmed dengue cases have been reported in US travelers returning from Brazil, Cuba, the Dominican Republic, Ecuador, Haiti, Jamaica, and Puerto Rico.Middle EastDengue activity is reported occasionally throughout the Middle East, including areas popular with travelers, such as Jeddah in Saudi Arabia. Currently, dengue cases are being reported in Pakistan and Yemen.Source:
23The Global Resurgence of Dengue Unprecedented global population growthUnplanned and uncontrolled urbanizationLack of effective mosquito vector controlGlobalization of tradeMore man-made breeding grounds (waste)Increased international air travelDecay in public health infrastructureAedes aegypti in the Americas (1970), at the end of the mosquito eradication program, & in 2002Emerging infectious diseaseIncidence on the rise globallySCARY GRAPHDengue in central and south america resurgence
28Dengue in the USA Some historical dengue outbreaks in the USA 1780: Philadelphia, PA1826-8: Savannah, GA1850-1: Charleston, SC, Savannah, GA, New Orleans, LA, Mobile, AL, Galveston, TX, Augusta, GA1922: Texas, Savannah, GA1934: Florida1945: New OrleansThe first indigenous transmission of dengue in the United States since the 1940s occurred in Texas in 1980.Other recent clusters in the United States also occurred in Texas, where 25 locally acquired cases occurred inA dengue outbreak occurred in Hawaii during In this case Aedes albopictus mosquitoes were responsible for virus transmission. in homes.Conditions favorable to dengue transmission exist in the southern US, and bursts of DF activity are to be expected in this region, particularlt along the Mexican border, where water may be stored in containers and A.aegypti numbers may be the greatest.
29Dengue in the USA (2) Recent indigenous transmission Texas: Hawaii: 1980: 23 cases, first locally acquired since 1945: 64 cases (lab-documented)2005: DEN-2 epidemic in Brownsville; estimated incidence of recent dengue infection (4% of population)Hawaii:: 122 cases (first since 1944)Florida (Key West):: 93 cases (as of 17 May 2011)6 cases to date in 2011: Miami-Dade (2), Palm beach (2), Martin (1), Hillsborough (1)1 Counties1Anil L, Stanek D, Blackmore C, Stark L, Mock V.
30Travel-Associated Dengue ArboNET and CDCDB databasesSera from 732 US travelers264 confirmed (36%)468 probable (64%)History of travel (649, 89%)263 (43%) Caribbean; 208 (34%) Mexico, Central/South America;131 (21%) Asia, Pacific Islands;12 (2%) Africa.ArboNET (596 cases)429 (72%) uncomplicated DF, 95 (16%) DHF/DSS, 56 (9%) Other, 16 (3%) Dengue with hemorrhageCDCDB (136 cases, syndromes available for 54 (40%)32 (59%) uncomplicated DF, 4 (7%) DHFSource:
32It is here! Dengue fever outbreak feared in Key West [Updated] July 14, 2010|By Thomas H. Maugh II, Los Angeles TimesFederal officials said Tuesday that they fear an outbreak of dengue fever in Florida after a survey of Key West residents found that at least 5% had been infected or exposed to the virus. With the exception of a handful of isolated cases along the Texas-Mexico border, there had previously been no cases in the continental United States since 1946 and no outbreak in Florida since 1934.5% of Key West Population Infected in 2009;New Case Suggests Ongoing OutbreakBy Daniel J. DeNoon WebMD Health NewsReviewed by Laura J. Martin, MDAlthough only 28 cases have been definitively identified, blood tests conducted in September 2009 detected evidence of recent infection in 5.4% of 240 randomly selected residents.The latest case of the mosquito-borne disease was in mid-April. It's not yet clear whether the April case is a continuation of the 2009 outbreak or a new outbreak from a different dengue strain.May 20, An "extended outbreak" of dengue fever is ongoing in Key West, Fla., where some 5% of residents were infected last fall."The best estimate from the survey is that about 5% of the population of Key West was infected in 2009 with dengue," dengue expert Christopher J. Gregory, MD, of the CDC's Epidemic Intelligence Service, tells WebMD.
33Key West DengueRT-PCR done on 1,178 pools of Ae. Aegypti mosquitoes collected from Monroe County, FL from 27 January-17 December 2010DENV-1KW sequence grouped as a member of a large clade of recent DV from Central AmericaNicaragua, 2006, 2008Unknown time of introduction into FLGraham AS, Pruszynski CA, Hribar LJ et al., Emerging Infectious Diseases • • Vol. 17, No. 11, November 2011
35Dengue Impact on U.S. Military Operations CubaPhilippinesWorld War IIVietnamDengue has always had an adverse impact on military operationsLikelyHospitalized US military personnel, Philipines
36Pre-WWII Cuba Panama Philippines 1897, exact # cases unknown Priority for YF and typhoidPanama1904, 200 casesPhilippines1906 outbreak at Ft. William McKinley: 101 per 1,000 persons/year (7 days)40% of new arrivals acquired dengue within the first year; 30% had recurrent illness, and 15% had a third episodeDengue was second to VD as the most common illness: 4,000 work days were lost each year
37WWII South Pacific: Australia: 80,000 hospitalized 1942:1600 hospitalizations (Changi, Singapore); “Almost all troops” (Tulagi)1943: Espiritu Santos, New Hebrides (5,000 affected (25%); Hawaii (Waikiki beach, resulting in travel restrictions)1944: Saipan (7 epidemics, 3500/1000/yr), controlled vector by spraying (148th General Hospital, 176th Station Hospital)1945: Okinawa (275/1000/yr): New Guinea/Philippines (27,000 cases)China-Burma-India: 8,000 hospitalized: 40/48 US troops developed DF within 5-10 days of arrival.Australia:1942: 80% of service members were affected.1943: 463 cases
38Vietnam War 1964: Ubol, Thailand 58/294 confirmed DF 1966: 31/110 DF among FUO cases: 8th Field Hospital 10/94 (11%)1967: Mekong River Delta 3%: average monthly incidence of DF (3.5/1000)3-28% of FUO cases were dengue
39Post Vietnam War Somalia Haiti Operation Restore Hope (1993, 30,000 troops)129/289 (45%) did not have an immediately identified cause of illness; 59/96 (61%) had laboratory-confirmed DF69/289 (24%) suspected DF casesHaitiOperation Uphold Democracy (1994, 20,000 troops)30/103 (29%) hospitalized febrile troops had DFFollow-up UN mission: 79/249 (32%) had DF (86th CSH)
40Present Time Defense Medical Surveillance System (DMSS) : 26 DF cases hospitalized, 170 ambulatory: 9 SOF soldiers developed DF: 97 dengue cases (47 Army) reported among active duty personnel of the US DoD
41USASOC Study I Seroprevalence study USASOC personnel deployed to dengue-endemic areas in Latin AmericaAt least 30 days, from500 specimensDoD Serum RepositorySandwich ELISA48/500 (9.6%) seroprevalence rateCaci JB, Lyons AG, Tack DM; ASTMH Abstract # 90, 2010
42USASOC Study II Seroconversion study Initiated 2012 Ongoing USASOC personnel deployed to dengue-endemic areas in Latin America, SE AsiaPre- and post-deployment seraSandwich ELISAPre-deployment seraNot counted in Study I22/213 (10.3%) seroprevalence ratePost-deployment sera results pendingCaci JB, Blaylock JB, DeLa Barrera R, Lyons AG; ASTMH Poster # 1089, 2012
44PathogenesisMultiple theories regarding pathogenesis but none acceptedLack of a reliable animal modelComplicated host and viral interactionsDifferent responses in adults and infantsAntibody dependent viral enhancementUpregulation of infectionIncreased cytokine activityUnknown etiology of capillary leak syndrome characterized by DSSBelieved that Aedes originated in jungles of Africa. Same vector as yellow fever and chikungunyaAlbopticus is Asian tiger mosquitoUses relatively fresh water from sources like water jars, vases, discarded containers, cocunut husks and old tires. It usually inhabits dwellings and bites during the day.
45Pathogenesis (2)No evidence of direct viral infection of endothelial cells1Transient disruption in the function of the endothelial glycocalyx layerA molecular sieveHypoalbuminemia, proteinuriaDENV and NS1 adhere to heparan sulfateIncreased urinary heparan sulfate excretion seen in kids with severe dengue21Leong AS et al. Semin Diagn Pathol 2007;24:227-362Avirutnan P et al. PLoS Pathog 2007;3(11):e183.
47Case 250 y/o man with multiple mosquito bites after exploring the Amazon during a recent (2 weeks ago) trip. Had been recently web surfing and found out about dengue. He asks you if he should take post-exposure prophylaxis against dengue. He has been asymptomatic. What do you do?A) Admit, put on ribavirinB) Reassure
48Clinical Manifestations Dengue Hemorrhagic FeverDengue ShockSyndromeThe incidence of dengue is likely very underreported. Only 50 to 90% of cases are symptomatic and only 10% of the total are actually reported. Dengue is a spectrum of disease ranging any where from no or mild symptoms to severe disease with DSS or dengue hemorrhagic fever.50 to 90% of casesDEN-2 and DEN-4Kyle J Annu Rev Microbiol :71-92.
49Clinical Dengue Spectrum of Clinical illness Primarily defined in Thai cohortsAsymptomatic infection %Undifferentiated FeverDengue FeverDengue Hemorrhagic Fever (DHF)/ Dengue Shock Syndrome (DSS)Case fatality rate for DHF <1% with proper medical management; >50% without.45%<5%
53Dengue Fever (DF) Incubation period 3-7 days Illness lasts ~7-10 days A range of clinical manifestationsThree phasesFebrileCriticalRecoveryKey is early recognitionTriage and management at lower echelons is critical to successful management
54DF (Febrile Phase) Lasts 2-7 days Abrupt onset high fever (≥38.5o C) 5-7 days fever (biphasic)RashEarly flushlike rash may be replaced by a macular/morbilliform rash. Late petechialArthralgias, myalgiasSevere headacheEye, Retro-orbital painLumbosacral pain
55DF (Febrile Phase II) Lab Anorexia, nausea, vomiting Sore throat, injected pharynx, conjunctivaRespiratory symptomsEpistaxis, gum bleeding, petechiaeClassic DF with some hemorrhage is NOT DHFMassive (GU, GI) rare.PE:FeverGeneralized rash (may be replaced by macular/morbilliform later on). Petechial lateRelative bradycardiaGeneralized lymphadenopathyHepatomegalyPositive tourniquet testLabProgressive decrease in WBC
59Case 327 y/o AD USMC from Puerto Rico presented with 2 days of increasing fever (>38.5 C), severe headache, rash, arthralgias, myalgias, while on deployment in the Philippines. After 5 days of illness, his fever suddenly resolved. Should you:1) Discharge2) Draw labs and observe
60Critical Phase I Occurs at time of defervescence Around days 3-7 of illnessTemperature drops to °C or belowLasts hoursSystemic vascular leak syndromeIncreasing hematocritHypoproteinemiaPleural effusionsAscites
61Critical Phase II Progressive leukopenia Rapid thrombocytopenia Degree of plasma leakage variesIncrease in Hct a good barometerShockIf is to occur, preceded by warning signsFollow fluid management closelyCXR, US
62Main Problems Shock from plasma leakage Organ Impairment Can have fluid overload secondary to overhydrationOrgan ImpairmentSevere hemorrhage
63Recovery PhaseAfter critical phase, hours of reabsorption of extravascular fluidWell-being, appetite improvesBradycardia commonHemodynamic status improvesGI symptoms abateBlood counts normalize (RBC>WBC>Plt)Diuresis occursProlonged convalescence
64Main Problems Hypervolemia Due to overly aggressive fluid resuscitation
66Severe Dengue I Severe plasma leakage Severe bleeding Shock (DSS)Serosal fluid accumulation with respiratory distressSevere bleedingClinically evidentMulti-organ involvementLiver: AST/ALT >1000CNS: Impaired consciousness, seizures, encephalopathyCV and other
67Severe Dengue II Clinical signs Tachycardia, peripheral vasoconstrictionDBP rises towards SBP (narrowed pulse pressure, ≤ 20 mmHg)Patient may be conscious and lucidPoor perfusionCold, clammy extremitiesRapid, weak pulseMental status changesSerosal fluid accumulation with respiratory distress
68Severe Dengue III Severe bleeding Multi-organ involvement Clinically evidentMulti-organ involvementLiver: AST/ALT >1000CNS: Impaired consciousness, seizures, encephalopathyCV (cardiomyopathy) and otherCoagulopathy
69Consider Severe Dengue Patient is coming from an area of high dengue risk, has had 2-7 days of fever, plus any of the following:There is evidence of plasma leakage, such as:high or progressively rising hematocrit;pleural effusions or ascites;circulatory compromise or shock (tachycardia, cold and clammy extremities,capillary refill time greater than three seconds, weak or undetectable pulse,narrow pulse pressure or, in late shock, unrecordable blood pressure).There is significant bleeding.There is an altered level of consciousness (lethargy or restlessness, coma, convulsions).There is severe gastrointestinal involvement (persistent vomiting, increasing or intense abdominal pain, jaundice).There is severe organ impairment (acute liver failure, acute renal failure, encephalopathy or encephalitis, or other unusual manifestations, cardiomyopathy) or other unusual manifestations.
70Risk Factors for DHF/DSS Pre-existing immunity from previous infection (heterogenous subtype)Diabetics, asthmatics, other chronic diseasesDENV typeDENV-1,3 > 2,4Increased time between infectionsUnder age 15Increased capillary fragilityHLA type and race*Caucasian>AAHLA Class-1 allelesFemale sexAB blood groupPromotor variant of DC-SIGN receptorSingle-nucleotide polymorphism in TNF gene*De la C Sierra B, Kouri G, Guzman MG. Arch. Virol., 2007, 152(3) Epub 2006 Nov. 16.
71Factors that reduce the risk of severe dengue RaceSecond or third degree malnutritionPolymorphisms in the Fc-gamma and Vitamin D receptor genes
73Case 430 y/o AD Sailor who recently returned from a TDY to Thailand 4 days ago. Has had 2 days of fever, excruciating HA, eye pain, severe myalgias, arthralgias, sweats and rash. You suspect dengue.How to diagnose?How to treat?
74Lab (Dengue Fever) Marked leukopenia Thrombocytopenia Moderate elevation of AST/ALTViral isolation to Day 5 onlyNegative malaria smearsDengue IgM (+) on Day 6 serumTakes 5 days to manifestPCR availableConvalescent: 4-fold rise in IgG may be required
75Lab (Severe Dengue)Positive tourniquet test (or hemorrhagic manifestations)Thrombocytopenia (<100,000)*Increase in aPTT, decrease in fibrinogenPlasma leakageHemoconcentration (Hct. inc. >20%)*Pleural effusion/ascitesPetechiaeHepatorenal shutdown with shockViral isolation from acute serumConvalescent IgM (+)Peak proteinuria**0.56 v g/d (P<0.001), onset 1 day after defervescence (-2 to 3 days)**Vasanwala FF et al. BMC Infect. Dis., 2011, Aug 5, 11(1): 212.
76Immune Response to Dengue: Antibody Specificity Increases over time NS-1: Effective days 1-5 post onset of symptomsIgM/IgG: Effective after day 5A diagnostic capable of detecting both is desirableAcuteAcuteConvalescentConvalescentDaySlide courtesy of Dr. Subhamoy Pal
77Sample Prep Collect 2 separate red gel separator tubes (“tiger-tops”) Gently invert 5 timesAllow blood to clot min. 30 mins (vertical)Centrifuge at full speed ( G) for 10 minPipette off serum into separate cryovialsRefrigerate or ice bath (2-8°C, ELISA/PRNT)RT-PCR: 2-8°C for up to 6 hours (immediate RNA extraction possible) otherwise, -20°C for up to 14 days. Limit to one freeze-thaw cycle.Isolation: -80°C until ready for transport
78Lab (3) Most readily available diagnostic tests ELISA (serology) MAC-ELISA, IgG ELISACross reactive with malaria, lepto, old dengue infectionsIgM/IgG (Primary infection >1.2 (1/100 dilution), >1.4 (1/20 dilution; secondary infection if ratio is less) not standardizedIgA (peaks at Day 8 after fever; undetectable by Day 40; cant distinguish primary vs. secondary infections)PRNT, microneutralization (serology)More specificityResearch, vaccine workViral IsolationMost specific, but time and labor intensive, cant diff 1º and 2º infxnsHAIRequires paired (acute, convalescent) seraDoes not discriminate between infections by different flaviviruses
79Lab (4) Nucleic Acid Amplification (NAAT) Antigen Detection RT-PCR, Real Time RT-PCR, NASBABetter sensitivity (80-100%) than isolationStandardization? False positive results? Expensive (expertise and equipment)Cant differentiate between 1º and 2º infectionsAntigen DetectionNS1, E/M antigensAntigen capture ELISA, lateral flow antigen detection, NS1 IgM, IgG responses.Do not differentiate between the different serotypesNot as sensitive as isolation or RNA detection
80Lab (5) Primary infection Secondary infection IgM first to appear, at end of 3-5 day fever period (~50%), day 6-10 (93-99%), peak (2-3 weeks), undetectable by 2-3 mos.IgG appears at end of first week of illness, persists for >year (possibly for life).RT-PCR can provide a same-day diagnosis with a similar sensitivity to cultureSecondary infectionIgM typically LOWER titer than primary infection; false negatives have occurredIgG typically HIGHER titer than primary infection; may x-react with other flaviviruses (JE, YF, WN)
81Criteria for Highly Suggestive and Confirmed Dengue Infection Highly Suggestive (One of the following)IgM + in a single serum sampleIgG + in a single serum sample with HI titer ≥ 1280Confirmed (One of the following)PCR +Virus culture +IgM seroconversion in paired seraIgG seroconversion in paired sera or 4-fold IgG titer increase in paired seraAdapted from Dengue and Control (DENCO) study
83Tests Used for the Lab Diagnosis of Primary Dengue Infection Diagnostic WindowSample RequiredSample StorageTurnaround TimeFacilities/CostViremia (Culture)Acute Phase (before Day 5)Serum, plasma, PBMC, tissuesRefrigerate if <24 hrs, if >,-80°C1-2 weeksMosquito/cell cxBSL-2/3, Mol. Biol equip, $$$RT-PCR(sensitivity: %)Serum, plasma, blood, tissuesRefrigerate if <6 hrs, if >, -20°C1-2 daysBSL-2, molecular biology equip$$$Real-time RT-PCRIsothermal amplification (NASBA)SST or red top tubeVirus isolation in cell culture and detection by IFAUsed with IgG ELISA to differentiate primary from secondary infectionUsed with IgM ELISA to differentiate primary from secondary infection
84Tests Used for the Lab Diagnosis of Primary Dengue Infection (2) Diagnostic WindowSample RequiredSample StorageTurnaround TimeFacilities/CostAntigen Detection1-6 daysSerum, tissuesRefrigerate or frozen1 day or more (histological)ELISA facilities ($$), Histology facilities ($$$)IgM ELISADay 5 to –Day 90 post infectionSerum, plasma, bloodFrozen or refrigerated1-2 daysELISA facilities, $IgM Rapid Test30 minsNo additional supplies, $IgG (paired sera) by ELISA, HI or neutralizationAcute sera: 1-5 days; Convalescent: after 15 days7 days or moreELISA facilities, BSL-2 for neutralization, $
85Rapid Diagnostic Tests (RDT’s) Current RDT’sImportant for:Quick diagnosis (lab results take time and require labs)In resource-limited settingsAlerts a unit to ID threatsHelpful for triage during outbreaksCurtail geographic spread of infectious diseasesStability operations and infrastructure buildingWorldwide demand for better diagnostics to manage treatment and preventionFuture RDT’sSlide courtesy of Dr. Subhamoy Pal
86Current Rapid Diagnostic Technologies AgglutinationFlow throughLateral FlowIsothermal Nucleic Acid TestsSolid PhaseSlide courtesy of Dr. Subhamoy Pal
87Product Introduction #1: IgG/IgM Dengue Duo Cassette 10μL of serum, plasma, or whole blood15 minute (time to result)Wu et. al. CDLI 2000, pp10 uL of serum1.5 hoursSlide courtesy of Dr. Subhamoy Pal
88Product Introduction cont'd #2: NS-1/IgG/IgM Dengue Duo Cassette120μL of serum or plasma15 minute (time to result)Osorio et al. Virology Journal 2010, 7:361Slide courtesy of Dr. Subhamoy Pal
89Standard Diagnostics Dengue Duo (NS-1) RDT NS1 AgIgG/IgM Ab3 drops (110 μl) of plasma or serumfor early acute phase samples (day 1 ~5)10 μl of plasma or serum for early convalescence phase samples (after day 5 ~ 14)Ag/Ab levelDayNS1 AgIgMIgGAg/Ab levelDayNS1 AgIgMIgG123571012123571012Slide courtesy of Dr. Subhamoy Pal
90Interpretation Negative Primary Secondary Slide courtesy of Dr. Subhamoy Pal
91Why make a primary/secondary determination? The majority (>90%) of DHF/DSS cases are secondary infectionsOne 20 year longitudinal study suggests that among all DHF/DSS cases 9% are primary and 91% are secondary (Nisalak, A., et al.,. Am J Trop Med Hyg, (2): p )Overall, 2-4% of secondary infections proceed to severe dengue. Other risk factors also need to be considered. (Guzman, M.G., et al.,1997. Am J Epidemiol, (9): p ; discussion 804)Positive Predictive Value of secondary infection leading to DHF varies by region and attack rate.8% and 91% don’t add up to 100%Slide courtesy of Dr. Subhamoy Pal
92Commercially Available NS-1 Products Rapid TestsBio-Rad StripTMSD (Focus) BIOLINE Dengue NS1 AssaySD (Focus) BIOLINE Dengue Duo IgM/IgG/NS1 AssayPanbio Dengue Early RapidELISA formatPanbio Dengue Early PanE (2nd Generation)SD NS-1 Dengue Ag ELISABio-Rad PlateliaTM Dengue NS1 Ag(Evaluated by NMRC)Slide courtesy of Dr. Subhamoy Pal
94Summary of data Panbio Dengue IgM/IgG Duo Cassette RDT - Down-selected from among several Dengue RDT’s- Meets KSA and Attributes of draft CDD- Marketed overseas with record of stability- Ideal after day 5 post-onset of symptomsSD NS-1 Cassette RDT’s developed recently- Available NS-1 RDT’s comprehensively evaluated- Required for early diagnosis of dengue between day 0-7 post-onset of symptomsTogether, the two RDT’s can enable dengue diagnosis through all stages of infection to fulfill capability gap.Slide courtesy of Dr. Subhamoy Pal
95Advantages and limitations of different dengue diagnostic tests Viral isolation and identification• Confirmed infection• Specific• Identifies serotypes• Requires acute sample (0–5 days post onset)• Requires expertise and appropriate facilities• Takes more than 1 week• Does not differentiate between primary andsecondary infection• ExpensiveRNA detection• Sensitive and specific• Identifies serotype and genotype• Results in 24–48 hours• Potential false-positives owing to contamination• Requires expertise and expensive laboratoryequipment
96Advantages and limitations of different dengue diagnostic tests: Serology IgM or IgG seroconversion• Confirmed infection• Least expensive• Easy to perform• IgM levels can be low in secondary infections• Confirmation requires two or more serum samples• Can differentiate between primary and secondaryinfection*IgM detection (single sample)• Identifies probable dengue cases• Useful for surveillance, tracking outbreaksand monitoring effectiveness ofinterventions*Primary infection: IgM-positive and IgG-negative (if samples are taken before day 8–10); secondary infection: IgG should be higherthan 1,280 haemagglutination inhibition in convalescent serum.
97Advantages and limitations of different dengue diagnostic tests: Antigen Detection Clinical specimens (for example, usingblood in an NS1 assay)• Confirmed infection• Easy to perform• Less expensive than virus isolation or RNAdetection• Not as sensitive as virus isolation or RNA detectionTissues from fatal cases (for immunohistochemistry,for example)• Requires expertise in pathology
98Management 1st step: Is this dengue? Live in/travel to dengue endemic areaFever and 2 of the following:Anorexia/nauseaRashAches/painsWarning signsLeukopeniaTT positiveLaboratory confirmed dengue
99Tourniquet Test (TT)Positive in up to 50% of patients with classical dengue and almost all with DHFNon-specificProcedure:Inflate BP cuff halfway between systolic and diastolic BP for 5 minutesReleaseCount # petechiae in a Quarter-sized patch below the cuff>20 is positive
100Management (2) 2nd step: Warning Signs* present? Abdominal pain/tendernessPersistent vomitingClinical fluid accumulationMucosal bleedLethargy, restlessnessLiver enlargement >2cmLab: Increased Hct with rapid decrease in Plt*Requires strict observation and medical intervention
101Management (3) Classifications Severe Dengue Dengue without warning signsMay be sent homeDengue with warning signsIF Dengue is suspected, and has warning signs,Dengue with no warning signs, BUT has co-existing conditions or social circumstancesReferred for in-hospital careSevere DengueSevere plasma leakage with shock and/or fluid accumulation with respiratory distressSevere bleedingSevere organ impairmentRequire emergency treatment
102Management (4) Dengue without warning signs Patients who do not have warning signs ANDAble to tolerate PO fluidsPass urine q6HLab: CBC: if stable Hct, can be sent homeTreatment: Bed rest, PO intake, paracetamol 4GM max/dayMonitoring: daily review for disease progression:Decreasing WBCDefervescenceWarning signs (until out of critical period)If any of these occur, return to hospital immediately
103Management (5)Dengue without warning signs BUT has co-existing conditions or social circumstancesCo-existing conditions: pregnancy, infancy, old age, DM, renal failureSocial circumstances: living alone, distance challengeLab: CBCRx: PO fluids, if not tolerated, start IV (0.9 NS or RL at maintenance)Monitoring:Temperature patternI/O (Uop)Warning signsCBC (Hct, WBC, Plt)
104Management (6) Dengue with warning signs Lab: CBC Rx Obtain reference Hct before fluid treatmentIsotonic solutions (NS, RL) 5-7 mL/kg/hr for 1-2 hr, then 3-5 mL/kg/hr for 2-4 hr, then 2-3 mL/kg/hr or less per clinical responseReassess clinical status, Hct, fluid infusion ratesIf Hct same or min rise, 2-3 mL/kg/hr for 2-4 hrCrashing: increase rate to 5-10 mL/kg/hr for 1-2 hrReduce IVF gradually when rate of plasma leakage decreases towards the end of the critical phase: adequate Uop/fluid intake, Hct decreases below baseline value in a stable patientMonitoringVS, peripheral perfusion (q15min-1hr) until pt is out of critical phaseUop (4-6 hourly)Hct (pre- post IVF, then 6-12 hourly)GlucoseRenal, hepatic, coags, CNS…prn
105Management (7) Severe Dengue Lab: CBC, other organ function tests Compensated ShockIVF (isotonic crystalloid, 5-10 mL/kg/hr over 1 hr).ReassessIf improved: decrease IVF gradually to 5-7 mL/kg/hr for 1-2 hr, then to 3-5 mL/kg/hr for 2-4 hr, then to 2-3 mL/kg/hr for 2-4 hr, then further per hemodynamic statusIf unstable: Check Hct after first bolusHct increased/high (>50%): repeat second bolus of crystalloid at mL/kg/hr; improvement? Then decrease to 7-10 mL/kg/hr for 1-2 hrs, continue to wean…Hct decreases: Cross match and transfuse blood ASAP
106Management (8) Hypotensive shock IVF resuscitation with crystalloids or colloids at 20 mL/kg as a bolus for 15 minImproved?Crystalloid/colloid solution (10 mL/kg/hr) for 1 hr, taper graduallyUnstable?Review Hct taken prior to first bolusHCT low (<40% in children/adult females, <45% adult males): cross match and transfuse!HCT high (WRT baseline): change to IV colloids at mL/kg as a second bolus over 30 min to 1 hr, reassessImproving: decrease rate to 7-10 mL/kg/hr (1-2 hrs), then back to IV crystalloids and reduce rates as aboveUnstable: repeat HctDecreased: bleeding…T/C, transfuseIncreased/remains high (>50%): continue colloid (10-20 mL/kg) as a 3rd bolus (1 hr), reduce to 7-10 mL/kg/hr (1-2 hr), change to crystalloid and reduce rate as aboveHemorrhage: PRBC (5-10 mL/kg) or whole blood (10-20 mL/kg)
107Management (9)ShockPROMPT, judicious fluid resusctiationKeep to minimum required to support CV stabilityPREVENTIVE transfusions should be avoidedDesmopressin? IV gamma globulin? Steroids? Drugs (chloroquine, balapiravir, statins)? No evidence for efficacyBeware pulmonary edema: may need PPVDHF-DSS is the 3rd most common cause of ARDS in hospitalized children in Malaysia
109Dengue Prevention Prevention: There is no prophylactic drug for dengue There is no licensed vaccine to prevent dengue (yet)Reduce risk by use of personal protective measures (DEET, permethrin-treated uniforms, screened windows, mosquito netting) and local vector control (eliminate breeding sites, insecticides)New approaches to vector controlGenetically-altered male mosquitoesEmbryonic introduction of wolbachia into A. aegyptiJury’s out……..
111And now for something completely different… 31 y/o female recently returned from Singapore…Fever (39.5°C), nausea, myalgias, back pain, HA, bilateral conjunctivitis, severe bilateral arthralgias (shoulders, knees, ankles, elbows, wrists, fingers).Lab: Lymphopenia (0.6 G/L), AST 177 UI/L, ALT 116 UI/L, LDH 780 UI/L, Nl Bili, CRP 64 mg/L.Course: developed chronic distal arthritis and tenosynovitis, swelling of the joints without fluid accumulation.
113SummaryDengue is a significant threat to the US military and civilian populations in endemic areas.Recognize atypical presentations: maintain healthy suspicionMay not have high case fatality rates, but illness will significantly affect mission(s)Vaccine development is underway and is challengingWRAIR is a leader in developing dengue vaccinesSeveral candidate vaccines are in the pipelineClinical ReadoutsMN optimization (50% reduction) and testing
114Widespread dengue a real possibility “Most individuals in the United States are as little concerned about dengue fever as they were a decade ago about West Nile fever. That situation could change if dengue continues its expansion as one of the world’s most aggressive reemerging infections.”Widespread dengue a real possibilityIncreasing presence of Aedes albopictus (36 states)Reemerged in South and Central America, Caribbean, and Puerto RicoIncreased outbreaks in Texas and HawaiiVaccine is needed but far from being readyThe first indigenous transmission of dengue in the United States since the 1940s occurred in Texas in 1980.Other recent clusters in the United States also occurred in Texas, where 25 locally acquired cases occurred inA dengue outbreak occurred in Hawaii during In this case Aedes albopictus mosquitoes were responsible for virus transmission. in homes.Conditions favorable to dengue transmission exist in the southern US, and bursts of DF activity are to be expected in this region, particularlt along the Mexican border, where water may be stored in containers and A.aegypti numbers may be the greatest.Morens D and Fauci A. JAMA :
115References2009 Dengue Guidelines For Diagnosis, Treatment, Prevention and Control (http://whqlibdoc.who.int/publications/2009/ _eng.pdf)CDC Dengue webpage (http://www.cdc.gov/dengue/)
116 Arthur Lyons COL, FS, MC DiLorenzo Health Clinic Corridor 8, Room 130 Arthur LyonsCOL, FS, MCDiLorenzo Health ClinicCorridor 8, Room 1305801 Army PentagonThe PentagonWashington, DC 20301TEL:FAX:
118Old Definition of Dengue Hemorrhagic Fever Fever lasting 2-7 daysTendency to hemorrhagePositive tourniquet test (TT)Spontaneous bleedingPlatelet count <100X109 per litreEvidence of plasma leakIncreasing hematocritPleural effusions
120Dengue Hemorrhagic Fever (DHF) Onset as per classical dengueDamage to blood and lymph vesselsDefervescence followed (2-5 days) byAscites, abdominal painPleural effusionHemorrhagic manifestations (gum bleeding, phlebotomy bleeding) which may progress to shockCentral cyanosisDiaphoresisEpi: Exposure in dengue endemic region with possible previous dengue infection