1CLINICAL MICROBIOLOGYSERVICEVIRUS HUNTERS& RESPIRATORY TRACT DETECTIVESDR. PHYLLIS DELLA-LATTALEARNING OBJECTIVES• Be familiar with viruses commonly associated with adult and pediatric patients• Discuss methodologies available for the rapid detection of viral pathogens• Distinguish common causes of community versus hospital associated pneumoniaVIRUSESTHE LOW DOWN• OBLIGATE INTRACELLULAR ORGANISMS• REQUIRE METABOLICALLY ACTIVE CELLS FOR EFFICENT REPLICATION• THERE IS NO UNIVERSAL CELL LINE • CERTAIN VIRUSES CANNOT BE CULTURED IN TRADITIONAL CELL CULTURE MONOLAYERS9 ROTAVIRUS9 METAPNEUMOVIRUS9 HEPATITISVIROLOGY TESTINGTHE MYTHS• TURN AROUND TIME [TAT] IS TOO LONG• CAN’T TREAT A VIRUS• ACADEMIC PURSUIT• MINIMAL IMPACT ON PATIENT CAREBUSTING THE MYTHS• TAT CAN RANGE FROM MINUTES TO <72 HRS9 DIRECT AG DETECTION9 MOLECULAR ASSAYS• MANY ANTI-VIRAL AGENTS AVAILABLE• CLINICALLY RELEVANTINFORMATIONPREDICTING THE PATHOGEN• AGE9 PEDIATRICS9 ADULTS• SEASON9 ENTEROVIRUSES: SUMMER/FALL9 INFLUENZA & RSV: WINTER9 ROTAVIRUS: WINTER/SPRING• IMMUNE STATUS• GEOGRAPHY• SKIN9 HSV, VZV, ENTERO• EYE9 ADENO, HSV, VZV, ENTERO• CNS9 ENTERO, HSV, CMV, VZV• GI9 ROTA, ADENO, ENTERO• GENITAL9 HSVRESPIRATORY VIRUSES CLINICAL SYNDROMES• COMMON COLD9 RHINO, CORONA, ENTERO, PARA 1-3, ADENO• PHARYNGITIS9 ENTERO, ADENO, EBV, HSV• CROUP9 PARA 1 & 2, RSV• BRONCHIOLITIS9 RSV, PARA 3, INFLU A & B• PNEUMONIA9 RSV, PARA 1-3, INFLU A & B, ADENO, CMV,VZV2MENU OF METHODOLOGIES• ROUTINE CULTURE• SHELL VIAL CULTURE [more rapid]• RAPID ANTIGEN DETECTION9 Enzyme Immunoassay [EIA]• HSV, RSV, INFLUENZA A & B, ADENO 40/41, ROTAVIRUS9 Direct Fluorescent Antibody [DFA]• CMV, HSV, VZV, INFLUENZA A & B PARAINFLUENZA 1-3, RSV, ADENO• NUCLEIC ACID AMPLIFICATION9 REAL TIME POLYMERASE CHAIN REACTION [PCR]VIRAL CULTURE• TURNAROUND TIMES9ROUTINE CULTURE• INCUBATE FOR UP TO 6 WEEKS• AVERAGE TAT – 5 TO 10 DAYS9SHELL VIAL CULTURE• SPECIMENS ARE CENTRIFUGED ONTO CELLS TO FACILITATE RECEPTOR BINDING• RESULTS IN 1 – 5 DAYSCUMC TEST METHODS & TAT 7 DAYS2 DAYS4 HRSADENO7 DAYS72 HRS4 HRSHSV21 DAYSENTEROVIRUS4 WKS72 HRSCMV2 HRSADENO 40/41CULTURESHELL VIALDFAEIAVIRUSCUMC TEST METHODS & TAT15 DAYS4 HRS2 HRSRSV15 DAYS4 HRSPARAFLU 1,2,315 DAYS4 HRS2 HRSFLU A & B21 DAYS5 DAYS4 HRSVZV2 HRSROTAVIRUSCULTURESHELL VIALDFAEIAVIRUSPEDIATRIC CASE OCTOBER, 2003: A 3 MTH OLD INFANT PRESENTED TO THE PEDS ED A “CROUP-LIKE”ILLNESS WITH LOW-GRADE FEVER. THE CHILD DID NOT HAVE A RECENT TRAVEL HISTORY.WHAT IS THE DIFFERENTIAL DX ?• VIRAL INFECTION?• BACTERIAL INFECTION?• QUESTIONS TO CONSIDER9 TIME OF YEAR?9 SICK FAMILY MEMBERS?9 IMMUNE STATUS?9 FAMILY PETS?• TESTS ORDERED?• SPECIMENS COLLECTED?3PATIENT RESULTS• EIA 9 POSITIVE FOR FLU A9 NEGATIVE FOR RSV• DFA 9 POSITIVE FOR FLU A9 NEGATIVE FOR RSV• CULTURE9 POSITIVE FLU A9SENT TO CDC & WHO FOR SUBTYPING• 1st CASE IN NYC-OCT9 COLUMBIA PRESBY CHONY (2002, 2003)• 2004 & 2005 SEASONS BEGAN LATE (DECEMBER) • RELIABILITY OF EIA DEPENDS ON CIRCULATING STRAIN OF FLUEIAFLU A+B• Standard Membrane EIA• Steps: 10-12• Assay time: 15-20 min• Hands on time: 7 min• Specimen Storage: 2-8oC • Differentiates A & BEIA INFLUENZA A & B98%SPEC70-87%SENFlu B91%SPEC71-95%SENFlu ADFADIRECT ANTIGEN DETECTION• IMMUNOFLUORESCENCE• VARIABLE SENSITIVITY• CULTURE BACK-UP NECESSARY• REQUIRES 103 –106VIRUSES/ML• RELATIVELY RAPID 92 - 3 hrs• INTERPRETATION CAN BE SUBJECTIVEDFA INFLUENZA A & B 99%SPEC83%SENSFlu B95%SPEC84%SENSFlu AADEQUATE SPECIMEN FOR DFA 9> 200 CELLS/SLIDE920 CILIATED EPITHELIAL CELLSTHE ABCs OF INFLUENZA• 114,000 HOSPITALIZATIONS & 20,000 DEATHS/YR IN U.S.• TYPES B & C 9 ONLY HUMANS (C IS VERY RARE))• INFLUENZA A9 AQUATIC BIRDS ARE NATURAL HOSTS & SERVE AS RESERVOIRS9 INFECTS HUMANS, OTHER MAMMALS (SWINE, ETC.), & BIRDS9 PIGS PROPOSED AS “MIXING VESSELS” FOR GENETIC REASSORTMENT BETWEEN HUMAN & AVIAN FLU A4INFLUENZA SUBTYPES• INFLUENZA SUBTYPES BASED UPON SURFACE GLYCOPROTEINS9 Hemagglutinin Activity (HA) 9 Neuraminidase Activity (NA)• NA CLEAVES CELL MUCIN BARRIER & HA FUSES TO CELLS SIALIC ACID RESIDUES, ENABLING VIRAL ADSORPTION & PENETRATION• 15 HA & 9 NA SUBTYPES 9H1-H3 & N1-N2 CAUSE OF WIDESPREAD DISEASE IN HUMANSINFLUENZA• ANTIGENIC DRIFT 9Mutations in HA & NA9Occurs during viral replication• ANTIGENIC SHIFT 9Only occurs with Influenza A9Trading of RNA segments between animal & human strains• GENETIC REASSORTMENT BETWEEN SPECIES SPECIFIC VIRUSES HAS BEEN ASSOCIATED WITH PANDEMICSANTIGENIC DRIFTGRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN SUBTYPEH3N2 H3N2 H3N2 H3N21968 1975 1993 2004HONG KONG VICTORIA BEIJING FUJIANSMALL GENETIC CHANGES REQUIRE ANNUAL VACCINATIONANTIGENIC SHIFTSUDDEN COMPLETE ANTIGENIC CHANGE DUE TO HA AND/OR NA SUBTYPE SUBSTITUTIONH1N1 H2N2 H3N21918 1957 1968SPANISH ASIAN HONG KONGINTERSPECIES TRANSMISSIONFLU PANDEMICS 20THCENTURY1. “SPANISH FLU” (1918-1919)9 H1NI STRAIN9 KILLED 50 - 100 MILLION WORLD WIDE 500,000 U.S.9 VERY VIRULENT• CYTOKINE STORM IN 15- 45 YO9 GENETIC MATERIAL FROM 1918 BEING ANALYZED• CLOSELY RELATED TO SWINE VIRUSES – PIG TO HUMAN TRANSMISSION• GENE MUTATIONS OF AVIAN VIRUS – NOT REASSORTMENT2. “ASIAN FLU” (1957)9 H2N2 STRAIN 9 KILLED 1 MILLION GLOBALLY, 70,000 U.S.9 3 OF THE 8 RNA SEGMENTS WERE RELATED TO AVIAN INFLUENZA VIRUSES [REASSORTMENT]5FLU PANDEMICS 20THCENTURY3. “HONG KONG FLU” (1968)• H3N2 STRAIN• HA GENE SEGMENT – AVIAN ORIGIN• KILLED 40,000 U.S.• LOWER MORTALITY DUE TO HA-ONLY SHIFT, NOT NA• 2 DUCK-DERIVED GENES & 6 HUMANFLUFROM CHICKENS TO HUMANS1997 HONG KONG H5N1 INFLUENZA 18 CASES & 6 DEATHS9 INDEX CASE – 3 Y.O. BOY9 PATIENT DIED OF EXTENSIVE INFLUENZA PNEUMONIA COMPLICATED BY REYE’S SYNDROME9 FIRST DOCUMENTED OUTBREAK OF AVIAN INFLUENZA A VIRUS IN HUMANS9 ESTABLISHED THAT AVIAN INFLUENZA VIRUSES CAN INFECT HUMANS WITHOUT PASSAGE THROUGH INTERMEDIATE HOSTS9 ALL GENE SEGMENTS WERE AVIAN, WHICH PROBABLY LIMITED ITS PANDEMIC POTENTIALFLUFROM CHICKENS TO HUMANS• H5N1 HONG KONG9 2003, 1 DEATH• H7N7 NETHERLANDS9 20039 HIGHLY PATHOGENIC 9 INFECTED PIGS & HUMANS9 83 POULTRY WORKERS & FAMILY9 79 CONJUNCTIVITIS9 6 RESPIRATORY SYMPTOMS9 FIRST DEATH WITH THIS STRAIN9 FIRST REPORT OF H7N7 CAUSING RESPIRATORY SYMPTOMS IN HUMANS1st CASE OF HUMAN TO HUMAN TRANSMISSION 2004An 11-YR OLD GIRL IN THAILAND• DIED OF PNEUMONIA SEPT 8 (H5N1)• RESIDED WITH 32-YEAR AUNT