zika diagnosis

my name is jane basile,and i am a microbiologist with the centers for diseasecontrol and prevention at the division ofvector-borne diseases in fort collins, colorado. this video is intended asan aid to diagnostic testing of patients suspected tohave zika virus infection. the material is aimedtoward trained laboratorians who have previous experiencewith viral diagnosis. the format used here is thesame as that which the cdc uses

for chikungunya virus igmelisa, and other arboviruses. while the reagent specificsgiven in this video are for zika virus, appropriatelystandardized reagents can be substituted into this testfor other arboviral agents. the recent designationof zika mac-elisa for use under the fda emergencyuse authorization, or eua, requires that laboratories in the us performingthis test must be trained and proficient accordingto federal guidelines prior

to testing patient samples. information on this is givenin the reference section at the end of this video. laboratory diagnosis ofzika virus is best performed on very acute-phase specimensusing real-time rt-pcr. for rt-pcr, samples shouldbe obtained at 7 days or less after onset of symptoms. however, not all samples arecollected in the acute phase. serology consists ofigm-elisa (sometimes referred

to as mac-elisa) and confirmatoryplaque-reduction neutralization test. this instructional videodescribes the proper way to perform an igmelisa for zika virus, and covers technical details,titration of reagents, and interpretation of results. reagents used in the videoare those currently used by the cdc arboviraldiseases laboratory.

those which arecommercially-available may be purchased from othersources, and dilutions used in this video only pertain to the lot numberscurrently being used. titration may be necessarywhen substituting reagents and under your ownlaboratory conditions. currently there are nocommercially-available kits for laboratory testingof zika virus infections. because of the cross-reactivityseen among flaviviruses,

it is preferable,but not required that dengue should be testedfor concurrently with zika, and dengue igm elisa kitsare commercially available from multiple sources. confirmatory serologicaltesting, the details of which arebeyond the scope of this video, should always be performedusing zika and dengue viruses. a laboratory risk assessmentshould be conducted prior to using this method.

zika virus is a salslevel 2 agent, and thus, precautions must be taken inhandling clinical specimens. the most common specimen typesused in igm elisa are serum and cerebrospinal fluidand there is potential for these specimensto contain live virus, or other unrelatedetiologic agents. the reagents used in the cdc igmelisa are all non-infectious. non-fda-regulated laboratoriesmay use infectious antigen although this isnot recommended.

laboratory safety precautionsinclude the use of lab coats or gowns, gloves, eye protection and certified hepa-filteredbiosafety cabinets. it is recommended thatmanipulations of the specimens and plate-washing are performedin the biosafety cabinet, and that liquid waste isdecontaminated using bleach prior to disposal. in laboratories where acertified biosafety cabinet is not available, it is recommended

that specimens areheat-inactivated using a water bath or heat block set at56 â°c for 30 minutes, and if chikungunyavirus is suspected, at 56 â°c for 2 hours. this will not adverselyaffect the igm elisa results. further information on thesafe handling and transport of sals level 2 agentscan be found on page 33 of the cdc/nih publicationbiosafety in microbiological and biomedical laboratories,

5th edition at the linkshown on the screen. the correct interpretation ofresults from either molecular or serologic methods used forzika virus laboratory diagnosis, is heavily reliant on the informationobtained from the patient. at a minimum, you shouldobtain the following: place of residence placestraveled to in the past year and dates of travel dateof onset of symptoms. this information may be missing

if asymptomatic personsare being tested, in which case it should be noted that the individualis asymptomatic. date of collection ofeach sample prior history of flavivirus infection and whether the patienthas been vaccinated for yellow fever,and if so, when? further information can be found on the zika infectionpage of the cdc website.

serum is the best andmost common sample type for this testing, as it isgenerally easy to obtain and the sample needed issmall, leaving the remainder for confirmatorytesting if indicated. cerebrospinal fluid(csf) is also acceptable, but volumes needed are larger. plasma and other sampletypes have not been evaluated in the igm elisa. whole blood is unsuitablefor serology,

and very bloody serum maygive unreliable results. do not accept samples drawninto anticoagulant tubes. samples should beshipped on ice pack, and kept at 4 â°cprior to testing. if the sample was taken lessthan 7 days after onset, molecular testing shouldbe performed first. ideally, paired samplesshould be obtained, with the second sample beingdrawn 7-14 days after the first. in reality, this isoften not practical.

complete information on howto obtain appropriate samples for testing can be foundat the link on the screen. immunoglobulin m istypically the first antibody to be detected in acuteprimary zika virus infections, and it persists for weeksto months or longer. igm levels are generallyhigher in primary infections than in secondaryflavivirus infections, where the patient hasexperienced a previous flavivirus infection.

the zika igm elisa isa qualitative assay. because igg accountsfor the majority of the serum antibody fraction,the igm elisa is configured to select for igm, which maximizes thesensitivity of the assay. this is accomplished bycoating the microtiter plates with anti-human igm antibodywhich attaches to the igm in the serum, andigg is washed away. after serum addition, antigenis added which is the source

of specificity of the test. an anti-flavivirus horseradishperoxidase conjugate is used to detect the reactionsand a tmb substrate is used to develop the results. for the non-commercialcomponents of the test, us laboratories may contactcdc using the information given these may be made availableon a case-by-case basis and are subject torestrictions of the eua. for international labs,

please contact your nationalreference laboratory. you will need the following:automatic microplate washer, programmed for 5 washes of300 âµl/well plate reader with 450 nm filter, and printer refrigerator at 4 â°c incubator at 37 â°c calibrated pipettors (p10, p200, p1000, multichannel) microtiter plates, high binding (currently we use immulon 2hb flat-bottomed 96-well plates)

pipette tips (non-blocked) serum dilution tubes & stoppers reagent reservoirs (boats) ziploc bags or sealed plasticcontainers paper towels for details regarding reagents,please see the list referenced for laboratories covered by theeua only, sources of reagents, and all details regardingstorage and handling of these must be adhered toaccording to the eua protocol.

to begin the igm elisa, markthe locations of the samples on the plate using asharpie as shown here. all samples and controlsare tested in triplicate on both viral andnormal antigens. outside wells are typically notused, to avoid inconsistencies that tend to occurin these wells. a maximum of 8 test specimenscan be tested on a plate. a positive and a negativecontrol should be included on the plate.

reconstitute theanti-human igm which serves as the coating antibody,and store according to the manufacturer'sinstructions. here we have used 50% glycerol to reconstitute thecoating antibody. prepare the coatingantibody solution, where 4.5 ml is requiredper plate, plus a little extrafor pipetting. the anti-human igm usedhere is diluted 1:2000

in coating buffer. when the reactivity of thecoating antibody is unknown, the antibody should be titrated to determine the optimumdilution according to the method given atthe end of the video, using the manufacturer'sdilution recommendations as guidance. add 75 âµl perwell of diluted antibody to each of the 60 test wells.

cover the plates using an emptyplate or plate cover and place in a refrigerator at 4 â°cfor a minimum of overnight and for a maximum of 7 days. if multiple plates arebeing processed at once, it is ok to stack the plates. when you are readyto run the test, remove the coating solutionfrom the plates by dumping it into the sink and blottingthe plates on paper towels. a plate washer should notbe used for this step.

the blocking buffer is madeof 5% nonfat dry milk in pbs with 0.5% tween 20,and can be stored at 4 â°c for up to 3 weeks. block the unused bindingsites on the plate with 200 âµl perwell of blocking buffer, cover the plate, andleave to incubate at room temperaturefor 30 minutes. while the plates are blocking, prepare the testsamples and controls.

test samples are bestprepared the day of use, but can be preparedup to one day ahead of time and refrigerated. dilute the test serumsamples 1:400 in wash buffer by adding 1 âµl of serum to 400 âµlof wash buffer. the negative controlshould also be diluted to 1:400 in wash buffer. if cerebrospinal fluidis the test sample type,

it should be used undiluted orat a maximum dilution of 1:5, and a negative csf controlshould also be placed on the plate at the samedilution as the test specimen. due to volume restrictions,it is reasonable to test csf in a single well on viraland normal antigens. samples should be dilutedin a biosafety cabinet or should have been heatedinactivated at 56 â°c for 30 min-2h if a safetycabinet is unavailable. dilute the positivecontrol according

to a previous titration, where the resulting opticaldensity should be around 1.0. this is to avoid wastageof positive control. in the test shown here, we are using the flavivirusgroup-reactive chimeric humanized monoclonalantibody 6b6c-1 diluted at 1:3000 in wash buffer. reconstitution of the chimericpositive control is done using water and once rehydrated,the control may aliquoted

and stored at -70 â°c. upon defrosting, thealiquot may be kept at 4 â°c forup to 6 months. if zika-positive serum isused, it should be diluted to at least 1:400, accordingto a previous titration. wash the plate using a platewasher located inside a safety cabinet if at all possible. the waste collection vesselshould contain bleach according to your safety regulationsto allow for inactivation

of any pathogenicsubstances prior to disposal down the drain. use 5 washes of 300 âµlper well. for testing not covered under the eua, if a platewasher is unavailable and the samples havebeen heat inactivated, and the viral antigen isconfirmed to be inactivated, wells can be washed by handby pipetting 200 âµl of wash buffer intoeach well and dumping

out for a total of 3 washes. vortex or mix thediluted test samples and controls immediatelyprior to use. add 50 âµl of each serumsample to 6 wells per plate, and do the same for thepositive and negative controls. cover the plate and placeit in a sealed container with wet paper towels tokeep the environment humid, or use a ziploc bagwith paper towels. place in a 37 â°cincubator for 1 hour.

for the cdc zika viraland normal antigens, store the lyophilizedproducts at -15 to -25 âºc and rehydrate with 0.25ml of sterile water. aliquot and store at -15to -25 âºc for up to 1 year. if an aliquot is notentirely used up in one run, the remainder can berefrozen and used later. discard at the end of 1 year. do not store theantigens at 4 â°c as they will deterioraterapidly.

the antigens can be usedto detect igm caused by all known zika virus strains. the zika virus antigen hereis used at a 1:160 dilution in wash buffer, and normalantigen is always diluted the same as the viral antigen. for non-us labs, if theantigen in use is infectious, it should be handledin a biosafety cabinet. us labs should onlyuse inactivated or non-infectious antigen.

when using a new lot number ofantigen, refer to the material at the end of the video for information onhow to titrate it. wash the plate usingthe plate washer. vortex or mix theantigens before use, then add 50 âµlof the viral antigen to the sample wells of the evennumbered columns on the plate, which correspond to the leftside wells for each sample. add 50 âµlof normal antigen

to the odd-numbered columnson the plate, which correspond to the right sidewells for each sample. cover the plate and placein the humidified container. incubate the plateovernight at 4 â°c. this is an important step forthe sensitivity of the test. shortening the incubation,for instance by incubating at 37 â°c for 2 hours, will cause low positivesto be missed. the detection conjugateused in this test is made

from a flavivirus group-reactivemonoclonal antibody, 6b6c-1, which was made to st.louis encephalitis virus. the monoclonal antibodyis conjugated to horseradish peroxidaseenzyme. this product is availablecommercially and should be reconstitutedand stored according the product used in thisvideo was made at the cdc and after reconstitutionin water, it is kept in a foil-wrappedcontainer

at 4 â°c for up to 1 year. dilute the conjugatein blocking buffer. each plate requires 3ml plus a little extra. commercial productswill need to be titrated to determine theworking dilution, using known working dilutionsof the other test components. video footage of this procedureis given toward the end of this presentation,and also in a document the lot number of the conjugateused here is diluted 1:5000

in blocking buffer,which is 1.0 âµl in 5.0 ml blocking buffer. make this dilution shortlybefore use and do not store. wash the plates. add 50 âµl of dilutedconjugate to all 60 wells. cover and incubate the platein a humidified container at 37 â°c for 1 h. in themeantime take the tmb substrate out of the refrigerator andbring it to room temperature. there are many commercialsources

of tmb substrate available andthey should be used according here, we are usingneogen k-blue. wash the plate, and then turnit 180 degrees and wash again, for a total of 10 washes. this is to ensure evenwashing of the wells. blot the plate on paper towel toremove any excess wash buffer. add 75 âµl ofsubstrate to all 60 wells. if substrate is beingadded to multiple plates, care should be taken toavoid substrate contamination

from the pipette tips. if the substrate in thereagent reservoir turns blue, discard the substrate and replace the reservoirwith a clean one. place in a dark place such as adrawer, or under several layers of paper towel atroom temperature. plate covers are notnecessary at this point. set a timer for 10 minutes. turn on the platereader to warm up.

after 10 minutes, add 50âµl 1n sulfuric acid to each well to stopthe reactions. (non-eua labs can use 75âµl of tmb stop solution to all the wells instead.) also add stop solution to oneor more wells on the outer edge of the plate to use as blanks. in both cases, avoidmaking bubbles in the wells as this will affect the reading. if bubbles occur, use a smallsharp object to burst them.

wait one minute beforereading the results. read the plate at 450 nmand print out the results. in this example, the value ofthe blank has been removed, but is usually 0.04 or less. subtract the mean opticaldensity of the blank wells from all of the test wells, thendetermine if the test is valid. calculate the mean od ofthe negative control serum. if the mean of theoptical density of the negative control reactedon the viral antigen is less

than 0.2, you can proceed. if it is equal to or higherthan 0.2, it is recommended that you repeat the test. determine the p-over-n (p/n)of the positive control. this is done by takingthe mean optical density of the positive control serumreacted on viral antigen, then dividing the productby the mean optical density of the negative controlreacted on the viral antigen. if the p/n value isgreater than or equal to 2,

then the test is valid. if it is not, then thetest should be repeated. if a repeat testyields invalid results, the reagents mayrequire re-titration. on a valid plate, calculatethe mean optical densities for each test samplereacted on the viral antigen. divide the product bythe mean optical density of the negative serum controlreacted on the viral antigen. this is the p/n forthe test sample.

if the p/n is less than 2.0, theresult is considered negative. for any samples with p/ngreater than or equal to 2.0, the result needs to beexamined for background reaction to the normal antigen. to do this, divide themean optical density of the sample reactedon the viral antigen by the mean optical densityof the test sample reacted on the normal antigen. if the product is less than 2.0,

this indicates a nonspecificreaction to the normal antigen and the results areuninterpretable. if a background reaction is notpresent and the p/n is equal to or greater than 2.0but less than 3.0, the result is consideredequivocal. if the p/n is 3.0 or greater,it is considered positive for igm to the zika antigen. all positive, equivocal, and uninterpretable resultsshould be confirmed with prnt.

here are examples ofnegative, positive, and uninterpretable results. always examine theresults closely. if a sample has (for example)2 viral antigen reactions with low optical densities, andone that is clearly very high, this likely indicatesan inconsistent well and you should repeatthe sample. failure to do so mayresult in a false-positive and unnecessary confirmatorytesting.

any igm or other test resultsshould always be interpreted in the context of otherdiagnostic test results, other samples from the samepatient, dates of onset and collection, travelhistory and clinical picture. igm elisa is not consideredto be a quantitative test; therefore, positive andnegative are relevant, but the p/n's themselvesshould not be compared directly to one another. because of the inherentcross-reactivity

of anti-flavivirus antibodieswith flaviviral antigens, it is important toinvestigate any positive results for other flaviviruses suchas dengue using a confirmatory plaque-reductionneutralization test, before a diagnosis is made, and results should beconsidered together. a negative igm result on asample taken less than 7 days after onset of symptoms isnot conclusive and may be due to delayed igm production.

a second sample taken 7-14days later should be requested in the absence of apositive rt-pcr result. here are some examples of how serologic testresults can be interpreted, and all assume that nomolecular evidence is available. this is an example of aprimary zika infection. the zika igm is positiveand dengue igm is negative, and the same is true forthe neutralizing titers. cross-neutralizationtests (that is,

tests that are performedconcurrently for both acute and convalescentsamples if available, on all relevant viruses) areusually performed regardless of whether igm resultsare positive for only one of the viruses. if a prnt titer is less than 10,or negative, for one virus and greater than or equal to 10, or positive, for the other, then the positive prntresult is considered to be the infecting virus.

this is an example of thesignificant cross-reactivity that can occur withinthe flavivirus genus. both zika and dengue igmresults have high p/n values and the infecting virus cannotbe identified definitively using plaque-reductionneutralization tests (prnt), therefore a laboratory diagnosisof flavivirus is given. traditional interpretationswhere a 4-fold difference between positiveprnt titers are used to identify the infectingvirus have been abandoned,

due to the confusionwith anamnestic responses in secondary the infections. here, the interpretation of flavivirus infectionavoids errors in the absence ofrt-pcr evidence. here, there is igm toboth zika and dengue, where the p/n is higherto dengue than to zika. however, the prnt shows thatthere is a higher titer to zika. because the prnt titers arepositive for both viruses,

it is impossible to saywhich the infecting virus is; hence the interpretationof flavivirus. this also illustratesthe dangers of trying to compare p/n valueswhen both are positive. this is an example of what apast flavivirus infection might look like: negative igm resultsfor both zika and dengue, and positive prnttiters of equal value. one would usually notperform prnt on a sample with negative igm results.

here, neither igm elisa norprnt give positive results, indicating that this patient hasnot been infected at any time with either of these viruses. this is an example of where igm and prnt give opposinginformation. a second sample, taken 7-14 dayslater would be needed to attempt to give a laboratorydiagnosis, and even then, a result more informative thanthis might not be forthcoming. as a general rule, primaryinfections are fairly easy

to diagnose via laboratoryresults. those who have livedfor part of their lives in a dengue-endemic area and will likely have hadprior exposure to one or more flaviviruses,are much more difficult to give a definitivelaboratory diagnosis for. hence, the importance of clinical correlationcannot be stressed enough. when you receive reagentsfrom cdc, we recommend

that you try them out first atthe recommended dilution shown on the product insert, forinstance, if it says use up to 1:160, then useit first at 1:160. see how the test looksusing the positive and negative control serum. titrate reagents if neededone at a time after that. for labs purchasing commercialconjugate you will need to titrate the productbefore use in the test. this is also true for new lotnumbers of the other reagents,

and the following will serve asan example of how to do this. in a 96-well plate, mark off4 columns containing 6 rows. coat and block theplate according to the standard igm-elisainstructions. add 50 âµlof positive control at a normal working dilutionto the first two columns. add 50 âµl of negativecontrol at a dilution of 1:400 to the third and fourth columns. cover and incubate at37â° c for one hour.

wash the plate and add 50 âµl of zika antigen at the normal workingdilution - in this case 1:160 to the first and third columns. add 50 âµl of normalantigen at the same dilution as the zika antigen to thesecond and fourth columns. cover and incubate at4â° c overnight. here, the conjugate will bediluted down at 1:500, 1:1000, 1:2000, 1:4000, 1:8000 and1:16,000 add 1 âµl of conjugate to500 âµl of blocking buffer.

vortex. put 50 âµl of blocking bufferinto the bottom 5 rows. put 100 âµl of the1:500-diluted conjugate in each well of the top row. take 50 âµlout of the first row and transfer it tothe second row. mix 3-4 times. repeat these stepsdown to the final row. discard the final 50 âµlfrom the bottom row.

incubate the platefor 1 h at 37â° c. wash the plate 10 timesand add 75 âµl of substrate per well. place the plate inthe dark and incubate at room temperaturefor 10 minutes. add 50 âµl per wellof stop solution and read at 450 nm after1 minute. the goal is to choose a dilution where the positive control givesan od of between 1.0 and 0.8,

and concurrentlygives an od of less than 0.2 for the negative control(ideally 0.05-0.1). here we can see thatthe od of 1.0 lands between the 1:4000and 1:8000 dilution. for this particularproduct, we chose 1:5000 as the working dilution. you should always test theworking dilution using the standard format andthe control sera prior to using with patient samples.

thank you for watching thisvideo and for adding this test to your laboratorytesting repertoire. we at the cdc are committed topublic health and excellence in testing, which can be bestaccomplished with national and internationallaboratory support.

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