BiodefenseLaboratoryN Y S D O H New York State Department of Health Wadsworth Center Validation of...

Biodefense Biodefense LaboratoryLaboratory

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New York State Department of Health

Wadsworth Center

Validation of testing Validation of testing algorithms and collection kits algorithms and collection kits for orthopox testing utilizing for orthopox testing utilizing

Laboratory Response Laboratory Response Network (LRN) methodsNetwork (LRN) methods


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Wadsworth Center

Vaccination Campaign Vaccination Campaign 20032003

• Risk of Smallpox as a Risk of Smallpox as a BT agent “increased” BT agent “increased” pre-Iraq war.pre-Iraq war.

• Available vaccine Available vaccine from original from original stockpile.stockpile.

• First stage of First stage of vaccination campaign vaccination campaign began January 2003began January 2003


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Wadsworth Center

Tasks for Biodefense Tasks for Biodefense LabLab

• Rapid response clinical vaccinia Rule-InRapid response clinical vaccinia Rule-In• Rapid response clinical variola Rule-OutRapid response clinical variola Rule-Out• Analysis of environmental unknownsAnalysis of environmental unknowns


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Wadsworth Center

Vaccinia virus Rule-InVaccinia virus Rule-In

• Live virus vaccine. Live virus vaccine.

• Wadsworth Center testing required for Wadsworth Center testing required for adverse event monitoring, transmission, adverse event monitoring, transmission, and release of VIG or Cidofovir.and release of VIG or Cidofovir.

• Requires rapid turn-around (<24h).Requires rapid turn-around (<24h).

• Requires enhanced containment Requires enhanced containment measures.measures.


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Wadsworth Center

Guidelines for Laboratory Testing at the NYSDOH Guidelines for Laboratory Testing at the NYSDOH Wadsworth Center Biodefense Laboratory for:Wadsworth Center Biodefense Laboratory for:

1.1. Vaccination-associated adverse events (VAE) Vaccination-associated adverse events (VAE) monitoring and confirmation during the smallpox monitoring and confirmation during the smallpox vaccination program vaccination program

2. Evaluation of patients with fever and an acute,2. Evaluation of patients with fever and an acute, generalized vesicular or pustular rash (i.e., suspect generalized vesicular or pustular rash (i.e., suspect

smallpox).smallpox).•Developed based on CDC guidelines•Collaboration between Biodefense Laboratory, Clinical Virology Laboratory, NYS LRN, and the NYSDOH Div. of Epidemiology

Preparation of Protocols and Submission Guidelines


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Wadsworth Center

Lab PreparationLab Preparation

• Assays:Assays:– LRN: real-time PCR, EMLRN: real-time PCR, EM– WC: real-time PCR, EM, culture (non-variola only), WC: real-time PCR, EM, culture (non-variola only),

DFA in development, multiplex rtPCR in developmentDFA in development, multiplex rtPCR in development

• Staffing (24/7): Staffing (24/7): – Current team (4 people) vaccinated.Current team (4 people) vaccinated.– Additional 6-8 vaccinated staff to perform primary Additional 6-8 vaccinated staff to perform primary

testing and support. testing and support. – Separate BSL-3 lab for Orthopoxvirus testing.Separate BSL-3 lab for Orthopoxvirus testing.


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Wadsworth Center

Lab ReadinessLab Readiness

• Collection supply list and Collection supply list and kitskits– Collection materialsCollection materials– Packaging/transport Packaging/transport

materials (labels, materials (labels, bags, shipping bags, shipping container).container).

– Forms (test requisition Forms (test requisition form).form).

– Protocols (specimen Protocols (specimen collection, packaging, collection, packaging, transport).transport).


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Wadsworth Center

Orthopox Collection Orthopox Collection KitsKits

• Purpose: Used to collect clinical specimens Purpose: Used to collect clinical specimens from VAE patients or from moderate to high from VAE patients or from moderate to high risk smallpox cases.risk smallpox cases.

• Contents:Contents:– Viral Culturette SwabsViral Culturette Swabs– Microscope SlidesMicroscope Slides– Screw-capped TubesScrew-capped Tubes– IATA Certified Shippers, Containers & Gel PacksIATA Certified Shippers, Containers & Gel Packs– Collection Guidance DocumentCollection Guidance Document– Collection Supply ListCollection Supply List


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Wadsworth Center

Orthopox Accessory Orthopox Accessory KitsKits

• Purpose: To facilitate safe collection of Purpose: To facilitate safe collection of specimens at non-clinical sites and enhance specimens at non-clinical sites and enhance specimen quality.specimen quality.

• Contents:Contents:– Absorbent UnderpadsAbsorbent Underpads– Biohazard BagsBiohazard Bags– Safety ScalpelsSafety Scalpels– Alcohol WipesAlcohol Wipes– Alcohol Gel Hand CleanserAlcohol Gel Hand Cleanser– RulerRuler– Sharps Disposal ContainerSharps Disposal Container


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Wadsworth Center

Distribution of Collection Distribution of Collection KitsKits

• Distributed to Regional Epidemiology offices.Distributed to Regional Epidemiology offices.• Distributed to Regional Resource Centers.Distributed to Regional Resource Centers.• Distributed to all local health depts.Distributed to all local health depts.• Surplus kits assembled and ready to go in Surplus kits assembled and ready to go in

storage.storage.• Protocols and guidelines were distributed to all Protocols and guidelines were distributed to all

sentinel hospitals.sentinel hospitals.• Collection kits, accessory kits, protocols were Collection kits, accessory kits, protocols were

distributed through Div. of Epidemiology distributed through Div. of Epidemiology

•


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Wadsworth Center

Testing AlgorithmTesting Algorithm

• ScreeningScreening– Perform clinical evaluation (rash algorithm, history form).Perform clinical evaluation (rash algorithm, history form).– Contact County HD and Local Epidemiologist.Contact County HD and Local Epidemiologist.

• SubmissionSubmission– Contact State Epidemiology.Contact State Epidemiology.– Determine method of transport to the laboratory.Determine method of transport to the laboratory.– Collect samples (scabs, slides, wet swabs [SOPs in kit]).Collect samples (scabs, slides, wet swabs [SOPs in kit]).– Package samples [SOPs in kit].Package samples [SOPs in kit].– Contact WC lab (518-474-4177 or 518-465-9720) with ETA.Contact WC lab (518-474-4177 or 518-465-9720) with ETA.– Complete poxvirus testing submission form [included in kit].Complete poxvirus testing submission form [included in kit].

• Lab and Epi BT coordinators exchange info.Lab and Epi BT coordinators exchange info.


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Wadsworth Center

Vaccinia Adverse Vaccinia Adverse EventsEvents

• Vaccinated:Vaccinated:– 825 NYS (excluding NYC)825 NYS (excluding NYC)– 39,608 USA39,608 USA

• Adverse Events:Adverse Events:– 30 NYS30 NYS– 3 patient specimens sent to the laboratory3 patient specimens sent to the laboratory


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Wadsworth Center

BDL-03-01BDL-03-01

• 34 year old female 34 year old female vaccination volunteervaccination volunteer

• Distribution of small Distribution of small lesions over bodylesions over body

• Team deployed with Team deployed with collection kitcollection kit

• Samples put through Samples put through the Adverse Event the Adverse Event AlgorithmAlgorithm


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Wadsworth Center

LRN Sample LRN Sample PreparationPreparation

• Extraction for PCR from Touch PrepsExtraction for PCR from Touch Preps– ~2 hours using BioRad Aquapure Extraction Kit~2 hours using BioRad Aquapure Extraction Kit– ~2 hours to run rt-PCR on ABI 7000~2 hours to run rt-PCR on ABI 7000

• Tissue Culture and EM from CulturettesTissue Culture and EM from Culturettes– No extraction timeNo extraction time– Grid Prep for EM ~1.5hoursGrid Prep for EM ~1.5hours– Tissue Culture results can take days to weeksTissue Culture results can take days to weeks

• Scabs and blood are other potential sample Scabs and blood are other potential sample sourcessources


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Wadsworth Center

BDL-03-01 ResultsBDL-03-01 Results

Published in JCM 42:1373-1375 (2004)Published in JCM 42:1373-1375 (2004)

Result: Laboratory- Confirmed GeneralizedResult: Laboratory- Confirmed GeneralizedVaccinia in 6 hours Vaccinia in 6 hours


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Wadsworth Center

BDL-03-02BDL-03-02

• Not a Vaccination Not a Vaccination VolunteerVolunteer

• Recent travel to ArgentinaRecent travel to Argentina• Atypical rashAtypical rash• Negative for Orthopox, Negative for Orthopox,

Vaccinia and VZV by rt-Vaccinia and VZV by rt-PCR and EMPCR and EM

• Low priority of case and Low priority of case and high probability of topical high probability of topical skin infection – no further skin infection – no further testing requiredtesting required


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Wadsworth Center

Aug. 2003-Mystery VirusAug. 2003-Mystery Virus“Expect the Unexpected”“Expect the Unexpected”• Female-mid 20’s presented at a clinic with small bump 10-12 Female-mid 20’s presented at a clinic with small bump 10-12

days after sexual contact.days after sexual contact.• Urine culture and sensitivity performed and swab taken.Urine culture and sensitivity performed and swab taken.• Vaginal swab was negative for Herpes but did produce viral Vaginal swab was negative for Herpes but did produce viral

response (CPE).response (CPE).• Specimen sent to Clinical Virology Laboratory, Wadsworth Specimen sent to Clinical Virology Laboratory, Wadsworth

Center for identification.Center for identification.• The viral isolate tested negative for HSV 1 and 2 by both The viral isolate tested negative for HSV 1 and 2 by both

PCR and ELISA, negative for enterovirus by PCR and IFA, PCR and ELISA, negative for enterovirus by PCR and IFA, negative for adenovirus by IFA, and negative for VZV by negative for adenovirus by IFA, and negative for VZV by PCR. The viral isolate was also hem adsorption negative PCR. The viral isolate was also hem adsorption negative ruling out orthomyxo- and paramyxoviruses. ruling out orthomyxo- and paramyxoviruses.

• Viral isolate in cell culture was then sent to Biodefense Viral isolate in cell culture was then sent to Biodefense LaboratoryLaboratory


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Wadsworth Center

Mystery VirusMystery Virus• Cell supernatant sent to BD Cell supernatant sent to BD

LabLab• rtPCR Positive for Vaccinia rtPCR Positive for Vaccinia

& Orthopox& Orthopox• Negative for Variola, Negative for Variola,

Monkeypox & RabiesMonkeypox & Rabies• EM confirms Orthopox EM confirms Orthopox

infectioninfection• Further investigation reveals Further investigation reveals

sexual contact with a recent sexual contact with a recent military vaccineemilitary vaccinee

Published in JCM 42:5409-5411 (2004)Published in JCM 42:5409-5411 (2004)


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Preparedness for Preparedness for MonkeypoxMonkeypox

in NYSin NYS• In place:In place:

– Collection protocols & kits for vaccination-Collection protocols & kits for vaccination-associated adverse event testing distributed associated adverse event testing distributed through-out statethrough-out state

– Collection protocols have been validated by Collection protocols have been validated by real-world positivesreal-world positives

– LRN real-time PCR (rtPCR) assays for VZV & LRN real-time PCR (rtPCR) assays for VZV & pan-Orthopox available and validated.pan-Orthopox available and validated.

– In-house rtPCR assays specific for variola In-house rtPCR assays specific for variola (smallpox), vaccinia, pan-orthopox & VZV (smallpox), vaccinia, pan-orthopox & VZV viruses available and ‘validated’.viruses available and ‘validated’.


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Prairie Dogs in NYSPrairie Dogs in NYS• 13 euthanized13 euthanized• Necropsies done at NYS Necropsies done at NYS

Wadsworth LaboratoryWadsworth Laboratory• Organs collected:Organs collected:

Spleen, Heart, LiverSpleen, Heart, LiverSalivary glands, Salivary glands, Cervical lymph nodesCervical lymph nodes& Epiglottis& Epiglottis

• Organs from each prairie dog Organs from each prairie dog pooled & ground with a mortar pooled & ground with a mortar and pestleand pestle

• DNA extraction using BioRad DNA extraction using BioRad Aquapure tissue extraction kitAquapure tissue extraction kit

Result: Negative for OrthopoxvirusesResult: Negative for Orthopoxviruses


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Human Monkeypox (July Human Monkeypox (July 2003)2003)

• Two patients suspected of contact with Two patients suspected of contact with infected prairie dogs:infected prairie dogs:– Patient 1: Blood onlyPatient 1: Blood only– Patient 2: Blood, roof of lesion & touch prepPatient 2: Blood, roof of lesion & touch prep

Negative Result for both patientsNegative Result for both patients


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Wadsworth Center

ConclusionsConclusions• BT Preparedness does work to enhance public BT Preparedness does work to enhance public

health for non-BT events!health for non-BT events!• Critical to have collaboration of lab andCritical to have collaboration of lab and

epidemiology personnel!epidemiology personnel!• Having carefully thought-out and proven Having carefully thought-out and proven

algorithms and protocols yield higher quality algorithms and protocols yield higher quality specimens and aids in confirmation of specimens and aids in confirmation of epidemiological findingsepidemiological findings

• Can use past preparedness plans for responding Can use past preparedness plans for responding to unanticipated events IF flexibility built into to unanticipated events IF flexibility built into algorithms and SOPs.algorithms and SOPs.


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AcknowledgementsAcknowledgements• NYS LRNNYS LRN

• NYSDOH Division of EpidemiologyNYSDOH Division of Epidemiology

• Wadsworth Center Clinical Virology Wadsworth Center Clinical Virology LaboratoryLaboratory

• Wadsworth Center Rabies LaboratoryWadsworth Center Rabies Laboratory

• NYS Biodefense LaboratoryNYS Biodefense Laboratory

• Electron Microscopy Core (Bill Samsonoff)Electron Microscopy Core (Bill Samsonoff)

BiodefenseLaboratoryN Y S D O H New York State Department of Health Wadsworth Center Validation of...

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