1. Describe the Epidemiology, Clinical Manifestations and Symptoms Of

9/9/2016

1. Describe the epidemiology, clinical manifestations and symptoms of mosquito-borne viral diseases: Dengue, Chikagungya, Zika, West Nile Virus, Yellow Fever and the encephalitis viruses: St. Louis, Eastern Equine, Western Equine, and La Crosse. CM Lynda Britton, PhD, MT(ASCP) SM 2. Discuss the laboratory diagnosis of these viruses. 3. Solve case studies on these viruses.

• Arbovirus disease cases vary from year to year • May cycle over 3 to 4 years • Vary by • # of mosquitoes that spread the disease • Weather that affects breeding • Cause outbreaks in the U.S. (West Nile virus) • Found outside the U.S. and come primarily from people who travel to those areas (Zika virus) • Most do not yet have vaccines • Prevention depends on people preventing mosquito bites and eliminating breeding sites • Clinicians must have a high index of suspicion to diagnose and report them

• Aedes aegypti--Most common mosquito transmitting viral diseases • Transmits dengue, chikungunya, yellow fever and Zika. • • Aedes albopictus also spreads all four diseases and West Nile Virus • Ae. aegypti has high vectoral capacity--effective transmitter • Ae. albopictus, Asian tiger mosquito, can live in more temperate (cooler) climates • Ae. aegypti lives in close proximity to people and their homes • Ae. albopictus is less likely to live as close or spread disease • Both are more likely to be active and bite in the daytime

1 9/9/2016

• Eggs survive months in dried out tires • Vector for a small outbreak of dengue in • Blacklegged mosquito Hawaii in 2001 • Found in swamp areas • Canada to south Florida and west to Texas • Caribbean and Mexico • Main vector of eastern equine encephalitis virus • Preferred host is birds.

• Overwinter as adults and lay their eggs in spring • Also known as Ochlerotatus triseriatus • Live in caves or hollow trees to stay warm • North American tree hole mosquito • In warmer climates active year round • Vector for the La Crosse encephalitis virus • Thrive in stagnant water like rain barrels, flower pots, rain gutters, and storm drains. • Bite humans when enter buildings • Prefer bird blood • Vectors of the West Nile virus along with 62 others

• Small single-stranded enveloped RNA virus in the flaviviridae • 4 serotypes (DEN1 to 4) • More severe disease with serotypes 2 and 3 primarily found in Asia • Immunity is type specific • Bite of Aedes aegypti found worldwide in altitudes below 1000 m • Aedes albopictus, Aedes polynesiensis and several species of the Aedes scutellaris complex can also spread

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• >70% of the world population most at risk live in Asia and • Pacific region • Epidemic spreading • >2.5 billion people live in dengue endemic areas • 35% case fatality rates in • 400 million cases occur each year rural India, Indonesia and • Most rapidly spreading mosquito-borne viral disease Myanmar • Worldwide fatality rate • Incidence ↑ 30 times averages 1% • Moved from rural to urban areas • Kills ~25,000 people each year • • Largest number of cases in Americas

• In U.S. citizens who live in Puerto Rico, U.S. Virgin Islands, Samoa, and Guam • Imported from people who traveled to or immigrated from endemic areas • Most common cause of fever in travelers returning from tropical or subtropical areas of the Caribbean, Latin America and Asia

• 3 outbreaks of dengue transmitted by mosquitoes • 75% asymptomatic • Hawaii in 2001 • Symptoms 3-14 days after bite • Texas in 2005 on the Mexican border • Ist phase (febrile) 2-7 days of fever, headache, pain behind the eyes, myalgia and bone pain, rash • Key West, Florida in 2009 • Low WBC count and evidence of bleeding • 10,000 confirmed cases in Puerto Rico in 2007 • Petechiae •High transmission season August to November • Nose or gum bleeding •Low transmission season March until July • Mild illness can be treated with rest, plenty of fluids and acetaminophen but no aspirin • Secondary transmission rare

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• Follows symptomatic dengue after 1-2 days of • RT-PCR in blood during 1st 5 days of symptoms-- sensitivity deffervescence 80-90% ; specificity 95% • IgM detectable in 50% of cases by 3-5 days after onset of • Can be fatal symptoms, 80% after day 5, and 99% by day 10 then declines • Capillaries become permeable and plasma leaks to undetectable after 2-3 months into chest or abdomen • Immunoglobulin M antibody capture enzyme-linked • Abdominal pain, vomiting, thrombocytopenia, immunosorbant assay (MAC-ELISA) • breathing difficulties, and possibly severe bleeding Cross reactivity • Confirmed by plaque reducing neutralization test • ~1% of patients die --usually children • IgG detectable by end of week one until several months or • Convalescence over 4-7 days years later • Vaccines are in development • Titer of 1:1280 considered highly suspect • Acute specimens drawn by day 5 and a convalescent specimen at day 10 or later required to demonstrate a 4-fold rise in titer

• Classical testing algorithms of dengue if molecular tests are negative: • 1. MAC ELISA • 2. IgG ELISA • 3. Plaque Reduction and Neutralization Test (PRNT)

• 61-year-old woman • Urgently needed • Sudden onset of left side weakness and unsteadiness • 5 candidate vaccines in clinical trials while walking • Dengvaxia, registered in Mexico in December 2015 • Lab tests normal except for a mild leukopenia • 3 doses to people age 9-45 years who live where (3500/mm3) and thrombocytopenia (70,000/mm3) dengue is endemic • MRI demonstrated cerebral ischemia • Younger patients and tourists are not eligible • Dengue fever diagnosed by RT-PCR • NIH has begun clinical trials on another live attenuated • ELISA positive for dengue II virus tetravalent vaccine that only requires one injection • 4 fold rise 3 weeks later • Reported effective in a small trial

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• Arbovirus in the genus Alphavirus in the Togavirdae family • Greatest in 1st week of infection --viremia high • Spread by Aedes aegypti and Aedes albopictus • Cases reported from mother to child • Humans reservoir host • In utero transmission may result in miscarriage or infection at birth • No cases of transmission from breast milk • No transmission through blood transfusion • A few laboratory-acquired cases

• Rarely seen in U.S. travelers before 2006 • 2006 to 2013 28 cases per year • Locally acquired in July 2014-- only 9 months after first cases reported in Caribbean • By January 2016, 679 cases reported from 44 states

• 2013 identified in local transmission in Caribbean countries and territories • Spread throughout Americas • 1.7 million cases

• 3-7 days after mosquito bite • 72-97% symptomatic • Lymphopenia, thrombocytopenia, elevated creatinine, • Fever and severe, debilitating joint pain and elevated liver transaminases • Rash, headache, nausea, vomiting, myalgia, and • Viral culture of blood in 1st 3 days (class 3 BSL conjunctivitis pathogen) • Most well in 7-10 days • RT-PCR in 1st week, and IgM antibodies after first 4 • Death rare days in serum or plasma • Most severe cases in people >70 years, with • Serological tests may not be positive until after first comorbidities, and newborns week of illness • Some have relapses of polyarthralgia, polyarthritis, • Confirmation by acute and convalescent plaque tenosynovitis, and/or Raynaud's syndrome months or reduction neutralization even years later

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• Woman in her early 30s • Recent travel to Haiti for a one week missionary trip • Single-stranded RNA virus • 3 days after returning: fever, myalgia and arthralgia in Flaviviridae family • Tests for dengue virus and chikungunya were positive for dengue IgG but negative for IgM antibodies • Most similar to dengue virus • IgM and IgG antibodies tests were negative for • Surface protein (glycoprotein-180) chikungunya virus helps flaviviruses attach to human cells • Diagnosed with dengue fever, put on bed rest and may explain how attacks nerve cells acetamenophen • Explains association with Guillain-Barre • After 3 weeks still had joint pain in hands and feet syndrome and birth defects • Repeat tests positive for chikungunya IgM and IgG • Aedes aegypti and sometimes Aedes • Dengue IgM and IgG antibodies negative albopictus • Demonstrates difficulty in diagnosing both chikungunya and dengue infections

• Locally acquired mosquito-borne cases reported: 35 • Travel-associated cases reported: 2,686 • Laboratory acquired cases reported: 1 • Total: 2,722 • Sexually transmitted: 23 • Guillain-Barré syndrome: 7

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• Found in sperm up to 93 days after symptoms began • Woman transmitted virus through vaginal fluid to man • AABB and FDA recommend all blood in U.S. be tested • 68 of 12,777 donors tested positive in Puerto Rico

• Most asymptomatic • Muscle weakness and possible paralysis • Few days-week • Form of acute motor axonal neuropathy • Rarely hospitalized • Most recover over time--Sometimes paralysis permanent • Patient's immune system attacks nerve cells • Sustained motor, low visual acuity and cognitive decline • Very rare cases of acute myelitis and meningoencephalitis • Acute disseminated encephalomyelitis rare • Damages white matter of brain • May last 6 months after Zika infection • Demyelination and polyfocal neurologic symptoms

• Arthrogryposis—joint • Miscarriage, stillbirth, severe contractures neurological damage • Musculoskeletal deformities • Brain damaged or stops growing • Secondary to neurological • Seizures, developmental delay, impairment intellectual disability • Arms and legs • Hearing loss-6%, movement disorders, • Malformations and difficulty feeding calcifications in cortex and • 7 cases of microcephaly; 5 pregnancies subcortical white matter lost since June 23 • Reduction in brain volume • Prior dengue infection, more severe Zika • Hypoplasia of the brainstem and cerebellum

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• Real time RT-PCR of serum, urine, CSF • ELISA IgM • Plaque-reducing neutralizing antibody test

• 33-year-old woman who visited Mexico, Guatamala and Belize • Do not travel to areas where Zika is spread with her husband • 11th week of pregnancy and remembered being bitten by • Prevent mosquito bites mosquitoes in Guatemala • WHO recommends none or protected sex for 6 months • Developed rash, ocular pain, myalgia, and mild fever lasted 5 days • Asymptomatic persons can spread the disease sexually • Husband also developed symptoms • Virus concentrated in semen and present long term • Positive for IgG and IgM antibodies to Zika virus ;1:2560 on PRNT • • Female to male transmission Between the 16th and 20th weeks of pregnancy • Fetal head circumference dropped to the 24th percentile • Abnormal intracranial anatomy. • Diffuse atrophy of cerebral mantal, shortened corpus callosum and loss of the frontal and parietal lobes of the cerebrum • Terminated pregnancy at 21 weeks • Fetus had high viral loads in brain—culture positive

• Most common cause of arboviral disease in the U.S. • 80% asymptomatic • Endemic in U.S. with periodic outbreaks

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• Blood transfusion, organ transplants • Mother to child: pregnancy, birth, breast milk • Laboratory workers • 63 species mosquitoes • 330 species birds • May to November

• 5,674 cases • 20-30% have fever, head and body aches, swollen lymph nodes, and/or rash • 51% presented with neuroivasive disease • <1% neuroinvasive disease • 56% encephalitis, • Meningitis—mild • 36% meningitis • Encephalitis—altered mental status, focal neurological • 8% flaccid paralysis defecits, tremors • Highest incidence 70 years • Flaccid paralysis—polio-like, limb paresis, respiratory and older paralysis • Guillain-Barre, cardiac arrythmias, hepatitis, • 9% died pancreatitis, orchitis, rhabdomylysis, and eye diseases • rare

• 70-year-old man • IgM antibodies in plasma or cerebral spinal fluid • 2-day history of headache and changes in mental status • Detectable 3-8 days after symptoms begin • CSF : 419 cells/µL, normal glucose and of 93 mg/dL • Persist for 90 days or up to a year in rare cases • Given antimicrobials for bacterial and herpetic meningitis. • Confirmed with plaque-reducing neutralization • 2 days after treatment began developed fever and needed • IgG positive requires acute and convalescent titers to be intubated demonstrating a four-fold rise • Admitted to ICU when developed left-sided weakness • Cultured and RT-PCR • WNV IgM positive 1:8 • Blood for donation tested by nucleic acid tests • Diagnosed encephalitis • Vaccine in development by NIH • Hospitalized for 42 days • After discharge residual left-sided weakness • Normal mental status

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• RNA virus: Togaviridae, genus Alphavirus • Endemic in North America • Only few cases each year in the U.S. • Spread by Culiseta melanura • Swampy areas near hardwood forests • People <15 years and >50 years • Atlantic, Great Lakes and Gulf Coast regions •

• Asymptomatic in 95% • Severe cases with a mortality rate of 33% • High fever, headache and vomiting • Disorientation, seizures and/or coma • Significant brain damage in survivors • 2012:15 cases • 5 died--median age of 76

• Pleocytosis with dominance of neutrophils and ↑ • RNA virus in protein CSF the Bunyaviridae • Difficult to culture • Transmitted by Aedes • RT-PCR of CSF and brain tissue triseriatus (the eastern • IgM of serum or CSF treehole mosquito)-- • Confirmed with acute and convalescent neutralizing aggressive daytime biter antibody

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• 5-15 days after bite • Fever, headache, nausea, vomiting, and lethargy • Severe neuroinvasive—seizure • Rarely fatal (about 1%) • Most fully recover

• Similar to other arboviruses • 19 cases of encephalitis in West Virginia in June • Viral encephalitis was the diagnosis in 11 • Serum or CSF IgM • 4 had meningitis • Confirmed by neutralizing antibodies • 4 had meningo-encephalitis • Age 1 to 14 years • Near large hardwood forest • 18 hospitalized • 1 death due to cerebral edema • 50% of cases patients have convulsions • 25% percent may have focal weakness or paralysis

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• RNA virus in the flaviviridae • Vectors are Culex species: • Birds are the reservoir hosts • Blue jays, robins, house sparrows and pigeons

• 99% asymptomatic • 5-15 days after the mosquito bite • Fever, headache, and mild meningitis • 90% of elderly develop encephalitis--confusion and disorientation, unsteadiness and tremors, and dizziness • Comatose • Rare death

Case fatality of 5-15%

• CSF -- lymphocytic pleocytosis and ↑ protein • Flavirus--single-stranded RNA virus • Difficult to culture • IgM immunoassay on serum or CSF • Confirmed by acute and convalescent IgG neutralizing antibody

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• In blood 3-6 days before symptoms begin & 5 days after • IgM and plaque neutralizing antibodies • Fever, severe headache, muscle pain, chills, anorexia, • Cultured if blood collected early enough in disease nausea, and vomiting • Vaccine--attenuated • Lasts 3-4 days and then most recover • Highly recommended, available many years, considered safe • ~15% jaundice, high fever, and bleeding and effective • In 10-14 days 20-50% of those with severe disease go into shock and organ failure and die • If they live, recovery may take many months • Mild yellow fever symptoms are similar to dengue and chikunguya; jaundice attributed to hepatitis •

• 47-year-old woman from Belgium who vacationed in Gambia • Did not receive a yellow fever vaccine because Gambia did not require it • 6th day after arriving in Gambia, became acutely ill with high fever, chills, headache, back and muscle pain • Returned to Belgium where she entered the hospital with jaundice, severe • Insect repellents should be used when outside asthenia (weakness), and anuria • Must contain at least 20% DEET and be EPA-registered • No neurological symptoms or bleeding but platelet count was 95,000/mm3, a BUN of 151 mg/dL, total bilirubin of 3.5 mg/dL, and greatly elevated • Higher percentage of DEET up to 50%, longer it lasts aspartate aminotransferase (49,000 U/L) • Hemodialysis was begun • Reapply when the directions say • Yellow fever was diagnosed by a positive RT-PCR. • Picaridin (KBR 3023, Bayrepel, and icaridin) Oil of lemon • Began bleeding from injection sites and her bowel. eucalyptus or para-menthane-diol, and IR3535 can also • Developed hypovolemic shock be used for mosquitoes but not ticks or bugs • Intubated and placed on ventilation • Received 4 units of fresh frozen plasma, 4 units of platelets and 2 units of • Sunscreen should be applied first and allowed to dry albumin • Products that contain both sunscreen and repellent are not • Remained in a coma & died 7 days after admission recommended by CDC

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• Wear clothing that covers your skin • Repellent applied by adults • Long-sleeved shirts, long pants, hats, socks and shoes • Do not get it on their hands—should not be • Treat clothing, tents, and other gear with permethrin. ingested • Can be purchased or applied (not skin) • Cannot be used on babies < 2 months of • Some mosquitoes bite through clothing age • Mosquito netting treated with permethrin • Drape mosquito net tightly over their carrier • 156 holes per square inch and be long enough to tuck under the or stroller mattress • Children under 3 years old cannot have Oil • Stay in lodging with air conditioning and screens on of lemon eucalyptus or para-menthane-diol windows and doors • Repellents safe for pregnant and nursing • Use mosquito coils containing metofluthrin or allethrin women

• Empty and scrub any item that hold water • Mosquito-proof rain barrels, septic tanks, plumbing pipes, gutters, and drains • Mosquito dunks for pond and fountains • Genetically modified mosquitoes approved in Florida by FDA • Aedes aegypti females will mate with the company's "OX513A" males, designed to produce offspring which do not fully mature