a.

i.
ii.

IDLTubextest:
TimetoPerform:1hourto1day
Description:Thenewtest(TUBEX)detectsantiSalmonellaO9
(bothimmunoglobulinM[IgM]andIgG)antibodiesinpatientsby
inhibitingthebindingbetweenanantiO9IgMmonoclonalantibody
(MAb)conjugatedtocoloredlatexparticlesandS.typhilipopolysaccharide
(LPS)conjugatedtomagneticlatexparticles(Limetal.,1998).Likethe
Widaltest,TUBEXconsistsofonestep,andtheresultisreadvisually
basedontheappearanceoftheliquidcontentsinthetube.Ontheother
hand,aspeciallydesignedsetoftubesisusedinTUBEX,and,insteadof
wholebacterialcells,S.typhiLPSadsorbedtomagneticparticlesisusedas
thedetectingreagent.Tomakethetestmorespecific,amonoclonal
antibody(MAb)whichrecognizestheimmunodominantO9determinantin
S.typhiLPSisalsoused;theantibodyisconjugatedtocoloredlatex
particles(Limetal.,1998).Whitelatexparticles(diameter,0.8um)were
sensitizedwithpurifiedantiO9antibodyorS.typhiLPSbypassive
adsorption(Limetal.,1998).Whentheantibodyconjugatedparticlesbind
totheantigensensitizedmagneticparticles,andthelatteraresedimented
byuseofamagnet,thecoloroftheliquid(supernatant)inthetubechanges
(Limetal.,1998).Whenmagneticparticlescoatedwithantigen(S.typhi
LPS)aremixedwithbluelatexparticlescoatedwithantiS.typhiLPS(O9)
antibody,thetwotypesofparticleswillbindtoeachother.Whenthe
magneticparticlesaresedimentedtothebottomofthetubebyuseofa
magnetattheendoftheexperiment,thebluelatexparticlesarealso
broughtdown.Thiswouldleavebehindaclearsupernatantifnotforthe
factthatcontrol(bovineserumalbumin(BSA)coated)redlatexparticles
arealsoaddedtothereactionmixtureandremainsuspendedinthesolution
throughouttheexperiment.Thismakesthesupernatantredwhentheblue
particlesaresedimented,whichiseasiertoseethanacolorlesssupernatant.
Whenapatient'santiO9antibodiesarepresentinthereactionmixture,
theywillinhibitthebindingoftheblueparticlestothemagneticparticles.
Consequently,thesupernatantremainspurplishblue(unchangedfromthe
beginning)duetothepresenceofblueparticlesandalso,inalower
concentration,ofredparticles(Limetal.,1998).Theinfectionspecific
antiO9antibodiesintyphoidpatientsaredetectedbytheirabilitytoblock
thebindingbetweenthetwotypesofparticles,hence,nochangeincolor.
TransformationoftheELISAtotheTUBEXtestwasmadepossiblebythe
demonstrationthatmagneticparticlescouldbeconvenientlyusedto
separatereactedfromunreactedindicatorlatexparticlesand,more
importantlyandmorerecently,bythedevelopmentofaspecialtubewhich
allowsefficientreactiontotakeplace(Limetal.,1998).Thetestkitkept
wellat4degreesC,asshownbythefactthatonlyaslightlossofactivity
wasdetectedafter9monthsofstorage(Limetal.,1998).Sensitivity6588
%,Specificity6389%(Bhutta,2006).Intheexaminationof16storedsera

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obtainedfrom14patientswithprovencasesoftyphoidfeverand78serum
samplesfrom75subjectswithouttyphoidfever,TUBEXwasfoundtobe
100%sensitiveand100%specific(Limetal.,1998).Noofsamplestested:
210,sensitivity26.9%,specificity88%(Nizamietal.,2006).
FalsePositive:Positivepredictivevalue24.1%(Nizamietal.,
2006).
FalseNegative:Negativepredictivevalue89.5%(Nizamietal.,
2006).
SerotypeTyphiIgMdipstickassay:
TimetoPerform:1hourto1day
Description:Thedipsticktest,developedintheNetherlands,is
basedonthebindingofS.TyphispecificIgMantibodiesinsamplestoS.
Typhilipopolysaccharide(LPS)antigenandthestainingofbound
antibodies,byanantihumanIgMantibody,conjugatedtocolloidaldye
particles(Ismail,2006).Serumsampleswerediluted(1/50)inthedetecting
reagent(containingdyelabeled,antihumanIgMantibodies).
NitrocellulosedipstickscoatedwithheatinactivatedserotypeTyphiwere
immersedinthedilutedserumandincubatedatroomtemperaturefor4h.
Thestripswerethenwashedanddriedatroomtemperature.Theserawere
graded(0to4)accordingtothestainingintensityofthecoloredband
correspondingtotheantigen(Houseetal.,2001).Sensitivity0.77and
specificity0.95(Houseetal.,2001).MultiTestDipSTicks:Approximate
cost(U.S.dollars)/specimen:10,No.oftests/kit:50;Amountofserum
needed10ul;Reactiontime90minutes;Temperatureforstorage28
degreesC(Olsenetal.,2004).
FalsePositive:PPV0.94(Houseetal.,2001).
FalseNegative:NPV0.80(Houseetal.,2001).
PanBioELISA:
TimetoPerform:1hourto1day
Description:ThePanBioutilisesadirectELISAformat.Briefly,
theSalmonellatyphiantigencoatedmicrowellstrips,dilutedabsorbed
samplesera,controlseraandcutoffcalibratorswereaddedandincubatedat
37degreesCfor20minutes.Residualserumwaswashed,andHRP
conjugatesantihumanIgMorantihumanIgGwasaddedandincubatedfor
another20minutesat37degreesC.Themicrowellswerewashedandthe
colourlesssubstratesystem,tetramethylbenzidine(TMB)andhydrogen
peroxide,wasaddedandincubatedatroomtemperaturefor10minutes.
Thereactionwasstoppedandthewellswerereadatawavelengthof450
mm(Gopalakrishnanetal.,2002).ThePanBioELISAkitsreflecteda78%
sensitivity,80%specificity(Gopalakrishnanetal.,2002).Comparsionof
thePanBioELISAwithresultsofcombinedTyphidotandTyphidotMtest
showedPanBioELISAtohavealowersensitivity,PPV,NPVandefficacy
butahigherspecificitythusreflectingontheantigenusedintheassay

iii.
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whichseemedtobemorespecificforS.typhi(Gopalakrishnanetal.,
2002).
FalsePositive:PPVof68.4%(Gopalakrishnanetal.,2002).
FalseNegative:NPVof87.4%(Gopalakrishnanetal.,2002).
IndirectSandwichELISA:
TimetoPerform:1hourto1day
Description:Inthisstudy,theserumantibodyresponsestothe
LPSandflagellumantigensofserotypeTyphiwereinvestigated(Houseet
al.,2001).InhouseindirectsandwichELISAswereestablishedtodetect
antiLPSIgA,IgM,andIgGandantiflagellumIgG(Houseetal.,2001).
Theserafromtyphoidpatientswere100%positiveforIgGantiprotein,
94.44%positiveforIgGantiLPSand88.89%positiveforIgMantiprotein
aswellasantiLPS.Incontrast,only61.11%and83.33%ofthesesera
gavepositiveWidaltestsforantiOandantiHantibodies(Mekaraetal.,
1990).Immulon1bflatbottom96wellmicrotiterplateswerecoated
overnightat4Cwith100ulofeither1ugofantigen/mlincoatingbuffer
(0.1Mcarbonatebuffer(pH9.4),antigenpositive)orcoatingbufferalone
(antigennegative).Theplateswereblockedfor1hat37Cwith200ulof
phosphatebufferedsalinecontaining1%bovineserumalbumin(BSA).
Serawereeitherassayedatasingledilution(1/1,000forantiLPSIgG,
1/500forantiLPSIgA,or1/250forantiLPSIgMandantiflagellumIgG)
orseriallydiluted(startingatadilutionof1/50).Seraweredilutedin
phosphatebufferedsalinecontaining0.1%BSAand0.05%Tween20,100
ulwasappliedtotheappropriatewells,andtheplateswereincubatedfor4
hatroomtemperature.Boundantibodies(IgA,IgG,orIgM)weredetected
usingheavychainspecificgoatantibodiesdirectlyconjugatedtoalkaline
phosphatase.Thelatterwerediluted(antiIgG,1/5,000;antiIgA,1/500;
andantiIgM,1/2,500)inTrisbufferedsalinecontaining0.1%BSAand
0.05%Tween20.Onehundredmicroliterswasaddedtoeachwell,andthe
plateswereincubatedovernightat4C.Onehundredmicrolitersofp
nitrophenylphosphate(1mg/ml)wasaddedtoeachwell,andtheplates
wereincubatedatambienttemperatureinthedarkfor30to40min.The
absorbanceat405nm(referencefilter,450nm)wasdeterminedusingan
automatedELISAreader(BioRad).Forseraassayedatasingledilution,
antibodylevelswereexpressedinopticaldensity(OD)units.Thesewere
takenasthemeanODofthreewellswithantigenminustheODofasingle
wellwithoutantigen.Forthetitrationassays,serawereassayedintriplicate
(twowellsantigenpositiveandonewellantigennegative),andthetiter
wastakenasthehighestdilutiongivinganetOD(meanODofantigen
positivewellsminusODofantigennegativewell)of>orequalto0.3
(antiLPSIgG)or>orequal0.2(allotherantibodies).Sixstandardswere
includedoneachplate,andtheODortiterofthesampleswasadjusted
accordingly.Blankwellswithnoserawereincludedtomonitor
background(Houseetal.,2001).Samplesweregradedas0to10according

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tothecolorofthereactionmixtureattheendoftheprocedure.Thosewith
agradeof>2wereconsideredpositive(Houseetal.,2001).
FalsePositive:Ataspecificityof[greaterorequalto]0.93,the
sensitivitiesofthedifferenttestswere0.75,0.55,and0.52fortheantiLPS
IgM,IgG,andIgAELISAs,respectively;0.28fortheantiflagellumIgG
ELISA(Houseetal.,2001).Amongindividualswithtyphoidfeverwho
hadpositivebloodculturesELISAtestswerepositivein89100%,whereas
theWidaltestispositiveinonly6183%(Mekaraetal.,1990).
FalseNegative:Amonghealthycontrols,7.517.5%hadpositive
Widaltestsbutonly05%werereactiveintheELISAassays(Mekaraet
al.,1990).TheWidaltestmaygivefalsenegativeresultsin1639%of
cases.Incomparison,therewerenofalsenegativeELISAtestsforIgG
antiproteinandonly6%forIgGantiLPS(Mekaraetal.,1990).
DiagnosticmethodfordetectionofSalmonellaentericaserovarTyphi
infecalspecimens:
TimetoPerform:1hourto1day
Description:Laboratorydiagnosisrequiresisolationand
identificationoftheorganismfromthepatient'sbloodorfeces(Vaishnavi
etal.,2006).IsolationofS.typhifromthefeces,bloodandotherclinical
specimensisthemostreliablemeansofconfirminganinfection.During
theincubationphaseofthedisease,thebacillimaybeoccasionally
cultivatedfromthefaecesandrarelyfromthebloodofthesocalled
'precociouscarrier'andsubclinicalcases(Vaishnavietal.,2006).Widal
testbasedonbacterialagglutinationhasremainedthemostwidelyusedtest
eventhoughitisneitherspecificnorsensitiveandtheresultcanbe
obtainedonlyafterthesecondweekwhenantibodiesareformed
(Vaishnavietal.,2006).FaecalspecimenswereinoculatedintoseleniteF
brothandincubatedat37degreesCfor6hoursorovernight(Vaishnaviet
al.,2006).Afterincubation,thebrothculturewascentrifugedat1000rpm
for10minutesforthedebristosettledown.Thesupernatantobtainedwas
checkedforthepresenceofViantigenwiththerespectivetestreagent.The
supernatantstestingpositivewithViantigenwereheatedfor1hourand
thencheckedforO9antigenandHdantigen.Thosesamplesnotgivinga
positivereactionwithViantigenwerecheckedwithoutheatingforO9
antigenandHdantigens.Strongagglutinationoccurringwithin1minute
wastakenaspositivefortherespectivetestreagent.Latexbeadscoated
withnormalrabbitantiserumconstitutedanegativecontrol.Allsamples
thatgavestronglypositivereactionswithatleasttwotestreagentswere
consideredtohaveS.typhi(Vaishnavietal.,2006).Diagnostictestsfor
typhoidfeveremployingantibodyresponsesmaynotcorrectlydetectthe
presentdiseaseasantibodyformationmaybedelayedorantibodiesmaybe
presentduetovaccinationormaybethereduetosubclinicalinfection
(Vaishnavietal.,2006).