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lntroduction
CuideIines foi lhe Ialoialoiy diagnosis of diphlheiia in
lhe WoiId HeaIlh Oiganizalion (WHO) Luiopean
Region
1
veie viillen in iesponse lo lhe ie-eneigence of
diphlheiia in easlein Luiope
1
. This docunenl appIies
lhe guideIines foi use in Ialoialoiies in lhe Uniled
Kingdon (UK).
The guideIines piesenl cuiienl ieconnendalions foi
lhe niciolioIogicaI diagnosis of infeclions caused ly
polenliaIIy loxigenic isoIales of coiynelacleiia, vilh
pailicuIai iefeience lo Ccrqncoac|criun ipn|ncriac and
C. u|ccrans. They covei lhe foIIoving nain aieas:
- Ialoialoiy safely issues
- lhe ioIe of lhe diagnoslic Ialoialoiy
- lhe ioIe of, and inleiaclion vilh, lhe iefeience
Ialoialoiy
- pioceduies foi piesunplive idenlificalion of
C. ipn|ncriac and C. u|ccrans
- inpoilance of loxigenicily lesling
- Ialoialoiy iesponsiliIily foi iepoiling loxigenic
C. ipn|ncriac, C. u|ccrans, and C. pscuc|uocrcu|csis
- suscepliliIily lesling of coiynefoin lacleiia
- seioIogicaI innunily lesling
- speciaIised lesling, noIecuIai lyping
- enhancing niciolioIogicaI suiveiIIance
Background
MicrobioIogy of infections caused by C. dphtheroe
and C. ulcerons
Diphlheiia is a iaie disease caused ly loxigenic sliains
of C. ipn|ncriac and, Iess oflen, C. u|ccrans. C. ipn|ncriac
is a Cian posilive, feinenlalive, pIeonoiphic iod, vhich
conpiises foui liolypes, vai gratis, vai ni|is, vai
in|crncius, and vai oc|fan|i. AII liolypes, vilh lhe
exceplion of lhe liolype oc|fan|i, nay pioduce lhe IelhaI
diphlheiia exoloxin. Diphlheiia is usuaIIy cIassified
accoiding lo ils sile of nanifeslalion, nosl cases aie eilhei
Laboratory guideIines for the diagnosis of
infections caused by Corynebocterum dphtheroe
and C. ulcerons
A Efstratiou, RC George
5unnary: Tncsc guic|incs rcprcscn| an app|ica|icn cf |nc lcr| Hca||n
Organiza|icn |urcpcan Rcgicns nanua| fcr |nc |aocra|crq iagncsis cf ipn|ncria
fcr |aocra|crics in |nc Uni|c Kingcn (UK), ou| |ncq ccu| oc app|ic |c |aocra|crics
ctcrscas. Tnc nanua| uas rcuri||cn in rcspcnsc |c |nc rc-cncrgcncc cf ipn|ncria
in cas|crn |urcpc an |nc cncrgcncc cf c|ncr infcc|icns causc oq Coiynelacleiiun
diphlheiiae an C. uIceians in |nc UK an ctcrscas. Tnc guic|incs sunnarisc
cur currcn| rcccnncna|icns an prcccurcs fcr |nc nicrcoic|cgica| iagncsis cf
infcc|icns causc oq |cxigcnic an ncn-|cxigcnic isc|a|cs cf ccrqncoac|cria, ui|n
par|icu|ar rcfcrcncc |c C. diphlheiiae an C. uIceians.
Ccmmun u|s Pu5||c Heo|th 1999: 2: 250-7.
A Lfslialiou, RC Ceoige
IHIS Respiialoiy and Syslenic Infeclion Ialoialoiy
Addiess foi coiiespondence:
Di AndiouIIa Lfslialiou
WHO CoIIaloialing Cenlie foi Diphlheiia and SlieplococcaI
Infeclions
Respiialoiy and Syslenic Infeclion Ialoialoiy
IHIS CenliaI IulIic HeaIlh Ialoialoiy
61 CoIindaIe Avenue
Iondon NW9 5HT
leI: O2O8 2OO 44OO, exl 427O
fax: O2O8 2O5 6528
enaiI: aefslialiouphIs.nhs.uk
TABLE 1 Toigenic C. dphtheroe isoIates referred to
5DRU: 1993 to 1998
Number ot Country 8iotype
Year isolates ot origin Source (var)
1993 4 Atrica skin m|t|s
Pakistan skin grov|s
Tunisia tbroat m|t|s
no travel* tbroat m|t|s
1994 4 8angladesb skin m|t|s
8angladesb tbroat m|t|s
|ndia tbroat m|t|s
Pakistan
|
tbroat grov|s
1995 1 Tbailand skin m|t|s
1996 3 Nepal skin |ntermed|us
Tbailand tbroat m|t|s
no travel* tbroat m|t|s
1997 3 eastern Lurope tbroat grov|s
|ndonesia skin m|t|s
no travel
|
tbroat grov|s
1998 3 Tanzania skin m|t|s
Tanzania tbroat m|t|s
Tanzania nose m|t|s
* contact witb overseas person
| tatal case ot dipbtberia
| laboratory acquired intection
Key words:
Corynebacteriun
infections
diphtheria
guideIines
Iaboratory infection
nicrobioIogicaI
techniques
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iespiialoiy oi culaneous. Mosl cases in lhe UK aie
liaveIIeis ieluining fion aieas vheie lhe disease is eilhei
endenic oi epidenic
2
. Toxin-pioducing sliains of
C. u|ccrans as veII as C. ipn|ncriac can cause iespiialoiy
diphlheiia, lul lhis is iaie
2,3
.
Lighleen isoIales of loxigenic C. ipn|ncriac veie
iefeiied lo lhe IHIS Slieplococcus and Diphlheiia
Refeience Unil (SDRU) fion vilhin lhe UK lelveen 1993
and 1998 (lalIe 1). One vas fion a 14 yeai oId loy vho
had liaveIIed fion Iakislan lo lhe UK and died of
diphlheiia
4
. Tvenly-lvo isoIales of loxigenic C. u|ccrans
veie aIso iefeiied lo SDRU, sone fion palienls noled
lo have a 'diphlheiilic nenliane. Ioui palienls
piesenled vilh infecled skin Iesions (lalIe 2).
Iion 1989 lo 1992, SDRU ieceived a lolaI of 95 non-
loxigenic isoIales of C. ipn|ncriac, nosl fion cases of
seveie and oflen iecuiienl lhioal infeclions
2,5
. Non-
loxigenic sliains of C. ipn|ncriac have sulsequenlIy
eneiged as polenliaI palhogens in lhe UK. The nunleis
of non-loxigenic C. ipn|ncriac isoIales iefeiied lo SDRU
(pailicuIaiIy lhe gratis liolype) iose fion 51 in 1993 lo
178 in 1997. In 1998, 147 oul of 163 non-loxigenic isoIales
veie of lhe liolype vai gratis, 15 vai ni|is, and one vai
oc|fan|i. Vai gratis is lhe piedoninanl liolype, isoIales
of vai ni|is aie connonIy isoIaled fion cases of culanous
infeclion, as seen eIsevheie in Luiope, in pailicuIai, in
aieas of easlein Luiope vheie lhe epidenic of diphlheiia
is decIining (T CIushkevich, I Mazuiova, peisonaI
connunicalion). This olseivalion has aIso leen nade
in olhei pails of Luiope. Il is nol cIeai vhelhei
innunisalion seIecliveIy inhilils giovlh of loxigenic
sliains oi vhelhei Ialoialoiies aie incieasingIy
idenlifying coiynelacleiia in diseases olhei lhan
iespiialoiy diphlheiia. Il has leen posluIaled lhal non-
loxigenic sliains occui noie fiequenlIy in peopIe vho
have leen innunised
6
. Non-loxigenic sliains have leen
iepoiled (aIleil iaieIy) lo cause syslenic disease, lolh
in lhe UK and oveiseas
2,7,8
, vhich nakes lhe appaienl
inciease in non-loxigenic liolypes of concein. The gIolaI
incidence of infeclions caused ly lhese oiganisns is
unknovn lul il is voilh iecoiding. The isoIalion of a
non-loxigenic isoIale fion a palienl vilh phaiyngilis
vilhoul lhe piesence of a nenliane is nol defined as a
case of diphlheiia
1,5
.
Ialoialoiy lased suiveiIIance of non-loxigenic
C. ipn|ncriac in LngIand and WaIes vas enhanced in 1995
and 1996
5
. IsoIales cane fion palienls in diffeienl
geogiaphic Iocalions and vilhoul any suggeslion of
inpoilalion fion oveiseas. The incieased nunlei of
infeclions since 199O couId le due lo inpioved
asceilainnenl, lul lheie has leen no upvaid liend in
lhe nunleis of loxigenic sliains in lhe UK. This
suggesls, lheiefoie, lhal lhe inciease in non-loxigenic
sliains is genuine
2
.
Characteristics of potentiaIIy toigenic
corynebacteria
The nininaI chaiacleiislics foi piesunplive
idenlificalion incIude posilive ieaclions foi calaIase,
niliale ieduclion (excepl lhe liolype oc|fan|i), cyslinase
pioduclion, and gIucose and naIlose feinenlalion and
negalive ieaclions foi uiease, suciose, xyIose, and
pyiazinanidase, vhich dislinguish lhe liolypes and lhe
olhei polenliaIIy loxigenic species (lalIe 3). The foui
liolypes of C. ipn|ncriac diffei liochenicaIIy: in
pailicuIai, vai in|crncius is IipophiIic, iequiiing Iipids
(foi exanpIe, hydioIysis on lveen 8O nediun) foi
oplinaI giovlh, as il giovs as a liny liansIucenl coIony
on iouline nedia. C. ipn|ncriac viII giov on nuliienl
agai, lul lhe piesence of seiun oi lIood viII inpiove
giovlh
6
.
5ignificance of Corynebocterum sp isoIates
Slandaid opeialing pioceduies foi lhe idenlificalion of
coiynelacleiia in diagnoslic Ialoialoiies aie leing
eslalIished ly lhe IHIS
9
. Il is advisalIe foi Ialoialoiies
lo eslalIish piolocoIs lo ensuie lhal appiopiiale aclion
is laken, hovevei, if coiynelacleiia aie isoIaled in lhe
foIIoving inslances:
- fion a iespiialoiy specinen, nolalIy lhioal and nose
svals
- in a vound oi skin sval/specinen fion a peison
vho has iecenlIy liaveIIed alioad
- as lhe piedoninanl oiganisn isoIaled fion a
noinaIIy sleiiIe sile, oi fion a vound, alscess, oi
puiuIenl spulun
- fion noie lhan lvo sels of lIood cuIluies
- al a IeveI 1O
4
coIony foining unils/nI in puie
cuIluie fion uiine
- fion nuIlipIe specinens
- as significanl giovlh of 'coiynefoins vilh vhile
lIood ceIIs in lhe oiiginaI Cian slain
TABLE 2 lsoIates of toigenic C. ulcerons referred to
5DRU: 1993 to 1998
Number ot Country Age range
Year isolates ot origin ot patients Source
1993 4 UK 9 to 54 years skin, tbroat (3)
1994 4* |taly 16 to 67 years nose, skin (2), tbroat
1995 4
|
Turkey 16 to 74 years tbroat
1996 5 UK 16 to 75 years skin, tbroat (4)
1997 4 UK 5 to 51 years tbroat
1998 1 UK 35 years tbroat
* one case associated witb ingestion ot unpasteurised dairy products in |taly
| toreign travel in Turkey
TABLE 3 BiochenicaI identification of cIinicaIIy significant
corynebacteria
Species CYS PYZ Nitrate Urea Glucose Maltose Sucrose Glycogen
C. d|phther|oe
var grov|s + - + - + + - +
var m|t|s + - + - + + - -
var |ntermed|us + - + - + + - -
var 5e|font| + - - - + + - -
C. u|cerons + - - + + + - +
C. pseudctu5ercu|cs|s + - - + + + - -
C. om,cc|otum - + v v + v v -
C. |m|tons - - - + + -
C. pseudcd|phther|t|cum - v + + - - - -
C. str|otum - + + - + - v -
: weak reaction
v: variable reaction
CYS: cystinase production on Tinsdale medium
PYZ: pyrazinamidase activity
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Laboratory safety issues
Ialoialoiy acquiied diphlheiia has leen iepoiled al
Ieasl lvice in lhe UK
1O,11
. To nininise iisks lo slaff, aII
cuIluies of suspecled C. ipn|ncriac and C. u|ccrans shouId
le handIed in a CIass 1 safely calinel. This is pailicuIaiIy
inpoilanl vhen piepaiing and handIing fIuid cuIluies
foi liochenicaI lesls. The pioduclion of aeiosoIs shouId
le nininised al aII lines and inhaIalion of aeiosoIs
shouId le avoided lo ieduce lhe iisk of diopIel
liansnission. Il is sonelines foigollen lhal lhe sliong
(NCTC 1O648) and veak (NCTC 3984) loxin pioducing
conlioI sliains used in lhe LIek lesl aie loxigenic, and
aie as IikeIy lo cause infeclion as cIinicaI isoIales.
In addilion lo safe handIing in lhe Ialoialoiy nalionaI
guideIines ieconnend lhal aII Ialoialoiy voikeis vho
nay le exposed lo diphlheiia in lhe couise of lheii voik
shouId le piolecled ly innunisalion
12
. A nininun
pioleclive IeveI of O.O1 InleinalionaI Unils pei nI (IU/
nI), as deleinined ly a loxin neuliaIisalion assay, is
ieconnended. Ialoialoiy slaff vho handIe loxigenic
sliains ieguIaiIy aie ieconnended lo nainlain lhe
highei IeveI of O.1 IU/nI. Ioi nosl aduIls in lhe UK
(vilh and vilhoul Ialoialoiy exposuie), vho aie
expecled lo have leen fuIIy innunised in chiIdhood, il
nay le noie appiopiiale lo loosl innunily and
exanine anlilody IeveIs al Ieasl lhiee nonlhs aflei
vaccinalion, ialhei lhan lesl, loosl if necessaiy, and ielesl
(as is lhe case anong Ialoialoiy slaff al lhe IHIS CenliaI
IulIic HeaIlh Ialoialoiy (CIHI)). Lxceplions lo lhis
geneiaI ieconnendalion vouId incIude lhose vho have
ieceived a looslei vilhin lhe pasl len yeais oi have had
an adveise ieaclion lo innunisalion. AddilionaI
loosleis shouId le given accoiding lo lhe iesuIls of
seioIogicaI lesling and individuaI iisks of encounleiing
loxigenic sliains.
RoIe of the diagnostic Iaboratory
The success of innunisalion in eiadicaling indigenous
diphlheiia in lhe UK and nosl deveIoped counliies has
Ied lo delale aloul lhe need lo scieen lhioal specinens
foi C. ipn|ncriac. The iaiily of cases in lhe UK, lhe
conpIex pioceduies associaled vilh Ialoialoiy
invesligalion, and lhe 'slieanIining of Ialoialoiy
pioceduies has Ied nany Ialoialoiies lo cease scieening.
Ialoialoiy expeilise in lhe idenlificalion of polenliaIIy
loxigenic coiynelacleiia has lheiefoie dininished. Since
lhe iesuigence of lhe disease in lhe Laslein Luiopean
Region, hovevei, lolh cIinicaI and niciolioIogicaI
avaieness has incieased in nany counliies and lhe
nunlei of pulIic heaIlh Ialoialoiies (IHIs) in LngIand
and WaIes scieening foi C. ipn|ncriac has incieased since
1997. The IHIS slandaid opeialing pioceduies (SOIs)
foi lhe exaninalion of lhioal specinens ieconnend lhal
aII lhioal specinens aie scieened foi C. ipn|ncriac
13
.
Sone IHIs have aIvays done lhis. In a fev Ialoialoiies,
vound svals - pailicuIaiIy fion palienls vho have
iecenlIy ieluined fion oveiseas - aie aIso scieened
ioulineIy.
In nany cases of iespiialoiy diphlheiia, cIinicaI
diagnosis piecedes niciolioIogicaI diagnosis, lul a
niciolioIogicaI iepoil of C. ipn|ncriac (and iaieIy,
C. u|ccrans) in iouline lhioal and olhei svals laken
fion lhe iespiialoiy liacl nay le lhe fiisl indicalion lhal
lhe disease shouId le consideied. In counliies Iike lhe
UK, vheie lhe disease is unconnon, lhe cIinicaI
diagnosis nay le difficuIl and nay le confused vilh
olhei condilions such as seveie slieplococcaI soie lhioal,
infeclious nononucIeosis, and Vincenls angina
2,14
. The
cIinician shouId lheiefoie le encouiaged lo excIude lhese
condilions. This highIighls lhe ioIe of lhe diagnoslic
Ialoialoiy in pioviding sinpIe, iapid, and ieIialIe
nelhods lo heIp cIinicians nake lhe coiiecl diagnosis
and lo eIininale suspecled cases oi conlacls of palienls
fion fuilhei conlioI neasuies and isoIalion.
RoIe of, and interaction with, the reference
Iaboratory
SDRU vas designaled a WHO CoIIaloialing Cenlie
foi Diphlheiia in 1998. The Ialoialoiy has cIose Iinks
vilh aII inleinalionaI iefeience cenlies, WHO, and
olhei nalionaI and inleinalionaI agencies. The nain
nalionaI ioIe of SDRU is lo piovide an 'on denand
iefeience seivice lo aII UK Ialoialoiies foi lhe
niciolioIogy of infeclions caused ly C. ipn|ncriac and
C. u|ccrans (lalIe 4).
Laboratory testing
Diagnoslic lesls used lo confiin infeclion incIude lhe
isoIalion of C. ipn|ncriac oi C. u|ccrans on piinaiy cuIluie
and loxigenicily lesling. Theie aie no definilive
conneiciaI lesls foi lhe diagnosis of diphlheiia, lul lheie
aie iapid lesls foi deleclion of loxigenicily, SDRU offeis
a diiecl poIyneiase chain ieaclion (ICR) and a iapid
enzyne innunoassay (LIA) on puie cuIluies lo confiin
loxigenicily in iesponse lo uigenl iefeiiaIs of isoIales.
The use of ICR as a piinaiy lesl on cIinicaI specinens
is sliII leing evaIualed and is nol cuiienlIy
ieconnended. Iuilhei delaiIs on lhe inleipielalion and
use of loxigenicily lesls aie given leIov (see |npcr|ancc
cf |cxigcnici|q |cs|ing).
CoIIection of cIinicaI specinens
MiciolioIogicaI cuIluie is essenliaI foi confiining
diphlheiia. A cIinicaI specinen shouId le ollained as
soon as possilIe vhen any foin of diphlheiia is
suspecled, even if liealnenl vilh anliliolics has aIieady
legun. Specinens shouId le laken fion lhe nose and
TABLE 4 PHL5 diphtheria reference service
|dentitication and toigenicity testing ot C. d|phther|oe and
otber potentially toigenic corynebacteria (C. u|cerons,
C. pseudctu5ercu|cs|s)
Dipbtberia immunity/vaccination studies
Antimicrobial susceptibility testing
Molecular typing
Training laboratory personnel in laboratory diagnostics tor
dipbtberia
Microbiological surveillance ot dipbtberia worldwide
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lhioal, and fion lhe nenliane, if piesenl. MuIlipIe sile
sanpIing shouId aIvays le consideied in a suspecled
case as lhis nay inciease lhe oiganisn iecoveiy iale and
haslen Ialoialoiy iesuIls. If possilIe, a specinen shouId
le laken fion undei lhe nenliane, vheie lacleiia aie
concenlialed
6,14
. RenovaI of seclions of nenliane foi
nicioscopy and cuIluie is aIso of vaIue. Nose and lhioal
specinens (usuaIIy svals) shouId le laken fion aII
suspecled cases and lheii cIose conlacls foi cuIluie.
IsoIalion of loxigenic C. ipn|ncriac fion cIose conlacls
nay heIp lo confiin lhe diagnosis of lhe case if lhe
palienls cuIluie is negalive.
Aflei coIIeclion, lhe svals shouId le senl lo lhe IocaI
Ialoialoiy innedialeIy, as iapid inocuIalion of speciaI
cuIluie nedia is inpoilanl. Al lhe sane line, lhe
cIinician shouId infoin lhe Ialoialoiy lhal diphlheiia is
suspecled.
The isoIalion of C. ipn|ncriac (usuaIIy non-loxigenic)
fion lIood cuIluies of palienls vilh endocaidilis fion
Luiope and AusliaIia aIso highIighls lhe inpoilance of
coiiecl isoIalion and scieening pioceduies foi lhese
oiganisns fion noinaIIy sleiiIe siles
7,8
.
Iioceduies foi lhe coIIeclion of specinens aie
desciiled fuIIy in lhe WHO ManuaI foi Ialoialoiy
diagnosis of diphlheiia
1
and aie sunnaiised in lalIe 5.
Transportation of specinens to the diagnostic
Iaboratory
If lhe lianspoilalion of specinens is IikeIy lo le deIayed,
lhe use of an appiopiiale lianspoil nediun - such as
lhe geI nediun desciiled ly Anies - shouId le
consideied
1
. If lhe liansil line viII exceed 24 houis,
lhe sval shouId le 'pieseived in a speciaI pack
conlaining a desiccanl such as siIica geI. Il is iepoiled
lhal C. ipn|ncriac can le isoIaled fion such svals aflei
deIays of up lo nine veeks
6,15
.
Mininun data required to acconpany specinens
The foIIoving dala aie inpoilanl cIinicaIIy and
epidenioIogicaIIy and shouId le coIIecled in lhe couise
of a case invesligalion.
- Ialienl delaiIs
nane, age, sex
hospilaI vheie leing liealed
allending physician
geneiaI piaclilioneis nane and conlacl delaiIs
- Ialoialoiy delaiIs
souice of specinen(s)
dale(s) coIIecled
line(s) of coIIeclion
- CIinicaI delaiIs
synplons
onsel dale
liealnenl (anliliolics, anliloxin)
dale liealnenl legan
dose and pIanned duialion of ieginen
- LpidenioIogy
suspecled case, conlacl, oi caiiiei
innunisalion hisloiy
liaveI hisloiy
delaiIs of any conlacl vilh fains, fain aninaIs,
oi unpasleuiised niIk oi niIk pioducls if
C. u|ccrans oi C. pscuc|uocrcu|csis infeclion
is suspecled
conlacl Iisl
5ubnission of C. dphtheroe isoIates to the
reference Iaboratory
AII UK isoIales of polenliaIIy loxigenic coiynelacleiia
(C. ipn|ncriac, C. u|ccrans, and C. pscuc|uocrcu|csis) fion
any lody sile (iespiialoiy oi culaneous) shouId le
iefeiied pionplIy lo SDRU (see addiess foi
coiiespondence, page 25O) foi confiinalion and
loxigenicily lesling. The unil offeis diagnoslic seivices
24 houis a day, seven days a veek, and nay le conlacled
al any line lhiough lhe CIHI svilchloaid (O2O8 2OO
44OO). Il is inpoilanl lo conlacl lhe unil lefoie isoIales
aiiive lo ensuie lhal lhey ieceive appiopiiale piioiily.
IsoIales shouId ideaIIy le sulnilled as puie cuIluies on
heaviIy inocuIaled lIood agai oi IoeffIeis agai sIopes,
sulnission shouId nol le deIayed vhiIe lhe sIope is
inculaled. In aII cases of suspecled diphlheiia il is
advisalIe lo liansfei isoIales lo lhe unil ly a couiiei.
Iiinaiy diagnoslic specinens aie nol usuaIIy
exanined ly SDRU, lul lhis is possilIe in sone
ciicunslances. Ioi exanpIe, in lhe fiisl inpoiled case
of diphlheiia fion lhe Laslein Luiopean Region, in May
1997, lhe diagnoslic Ialoialoiy iefeiied nenliane
sanpIes lo lhe unil, lecause lhey had nol al lhal line
isoIaled lhe causalive oiganisn fion nasophaiyngeaI
cuIluies
16
.
TABLE 5 CoIIection of cIinicaI specinens for the isoIation
of C. dphtheroe and C. ulcerons
Throat specinens
Tbe pbaryn sbould be clearly visible and well illuminated
Depress tbe tongue witb an applicator and swab tbe tbroat
witbout toucbing tbe tongue or inside tbe cbeeks
Pub vigorously over any membrane, wbite spots, or
intlamed areas, sligbt pressure witb rotating movement
must be applied to tbe swab
|t any membrane is present, litt tbe edge and swab beneatb
it to reacb tbe deeply located organisms
Transport tbe swab immediately to tbe laboratory tor
culture
NasopharyngeaI specinens
|nsert tbe swab into tbe nose tbrougb one nostril beyond
tbe anterior nares
Gently introduce tbe swab along tbe tloor ot tbe nasal
cavity, under tbe middle turbinate, until tbe pbaryngeal wall
is reacbed
Force must not be used to overcome any obstruction
Transportation to tbe laboratory immediately
5kin Iesions
Lesions sbould be cleansed witb sterile normal saline and
crusted material removed
Press tbe swab tirmly into tbe lesion
Transport to tbe laboratory witbout delay
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Prinary cuIture
Specinens shouId ideaIIy le inocuIaled onlo cuIluie
nedia vilhoul deIay. The diagnosis of diphlheiia lased
upon diiecl nicioscopy of a sneai is unadvisalIe as faIse
posilives and faIse negalives nay occui. The use of
seIeclive nedia, such as HoyIes TeIIuiile, is advised
1
.
The piinaiy cuIluie pioceduie is desciiled fuIIy in lhe
IHIS SOI foi lhe cuIluie of lhioal specinens
13
and in
lhe WHO ManuaI
16
.
Sone Ialoialoiies occasionaIIy use TinsdaIes
nediun foi piinaiy cuIluie, lul ils high seIeclivily
incieases lhe IikeIihood of faIse negalives, pailicuIaiIy
vilh specinens lhal conlain snaII nunleis of oiganisns
and il is lheiefoie nol ieconnended
1
. This cyslinase-
conlaining nediun is, hovevei, ideaI foi lhe
idenlificalion of piesunplive coiynelacleiia. Many
diagnoslic Ialoialoiies do nol slock such speciaI seIeclive
nedia as iouline piinaiy cuIluie on TinsdaIes nediun
foi C. ipn|ncriac is iaieIy undeilaken. In addilion, lhe
slaliIily and sheIf Iife of TinsdaIes nediun is shoil.
C. ipn|ncriac and C. u|ccrans viII aIso giov on slandaid
lIood agai and olhei piinaiy cuIluie nedia - foi
exanpIe, chocoIale agai.
The Ialoialoiy diagnosis of 'unsuspecled diphlheiia
infeclion can le difficuIl as coIonies of C. ipn|ncriac aie nol
dislincl upon lhe slandaid agai nedia used foi iouline
lhioal oi vound cuIluies. In addilion, lhe piesence of
'coiynefoins in nixed cuIluie vilh olhei lhioal oi skin
fIoia nay nol excile inleiesl unIess lhe Ialoialoiy is avaie
lhal lhe palienl iecenlIy liaveIIed oveiseas. Iuilheinoie,
lhe deleclion of C. ipn|ncriac as lhe causalive oiganisn of
culaneous diphlheiia oi olhei syslenic disease (foi
exanpIe, endocaidilis) is piinaiiIy ieIaled lo lhe fuII
idenlificalion of lhe 'coiynefoins isoIaled. Once isoIaled
il is easy lo idenlify C. ipn|ncriac oi C. u|ccrans.
Procedures for the presunptive
identification of C. dphtheroe and C. ulcerons
AII idenlificalion pioceduies aie desciiled fuIIy in lhe
WHO ManuaI and lhe IHIS SOI on lhe idenlificalion
of coiynelacleiia
1,9
. The pioceduie foi Ialoialoiy
diagnosis is oulIined in lhe figuie. The nininaI cuIluie
nedia iequiied aie lIood agai, leIIuiile lIood agai,
TinsdaIe agai nediun, and liochenicaI lesls foi
pyiazinanidase and, peihaps, uiease hydioIysis and
niliale ieduclion. The nininaI Ialoialoiy ciileiia
iequiied lo piesunpliveIy iepoil an isoIale as
C. ipn|ncriac and C. u|ccrans aie as foIIovs:
- calaIase posilive
- uiea: negalive foi C. ipn|ncriac, posilive foi
C. u|ccrans
- niliale posilive (excepl lhe liolype vai oc|fan|i)
- pyiazinanidase negalive
- cyslinase negalive
The lesls foi pyiazinanidase (IYZ) and cyslinase
(CYS) aie usefuI foi scieening in oidei lo dislinguish
lelveen lhe polenliaIIy loxigenic species and olhei
coiynefoins. If scieening lesls aie unavaiIalIe lhen
convenlionaI liochenicaI lesls shouId le enpIoyed -
foi exanpIe, uiease hydioIysis, niliale ieduclion, and
feinenlalion lesls foi gIucose, naIlose, gIycogen, oi
slaich. The liochenicaI chaiacleiislics of lhe polenliaIIy
loxigenic coiynelacleiia (foui liolypes of C. ipn|ncriac,
C. u|ccrans and C. pscuc|uocrcu|csis) and olhei
coiynefoins lhal nay le encounleied in lhioal oi vound
specinens aie desciiled in lalIe 3. The connonesl non-
loxin pioducing Ccrqncoac|cria spp. lhal nay le
encounleied aie C. anqcc|a|un, C. pscucipn|ncri|icun,
and C. s|ria|un
17
.
ConneiciaI idenlificalion kils idenlify C. ipn|ncriac
and C. u|ccrans vilh a good degiee of accuiacy. The AII
Coiyne (lioMeiiux, Iiance) vas lhe fiisl conneiciaI
kil. Il vas Iaunched in 1991 and is leing used
incieasingIy ly Ialoialoiies in lhe UK and oveiseas.
AIlhough ieIialIe foi lhe idenlificalion of C. ipn|ncriac,
lhe kil is sIighlIy coslIy lul is an exceIIenl aIleinalive lo
convenlionaI nelhods.
lnportance of toigenicity testing
The nosl inpoilanl lesl in lhe niciolioIogicaI
diagnosis of diphlheiia is lhe deleclion of loxin-
pioducing sliains. Toxigenicily lesls aie nol ieadiIy
avaiIalIe in nosl diagnoslic Ialoialoiies, il is sliongIy
ieconnended lhal aII isoIales le iefeiied pionplIy
lo SDRU, vhich is pioficienl in peifoining lhese lesls.
SeveiaI in vilio nelhods aie avaiIalIe al SDRU,
incIuding lhe convenlionaI LIek lesl, nodified LIek
lesl, a iapid LIA, and a genolypic lesl lased on ICR
foi deleclion of lhe 'A poilion of lhe loxin gene
18-2O
.
The nodified and convenlionaI LIek lesls aie used
ioulineIy in conjunclion vilh lhe LIA and usuaIIy yieId
iesuIls in lelveen lhiee and 24 houis. ecause of lhe
deIays in ollaining iesuIls, lhe seveiily Iinil upon
aninaIs, and lhe conpIex naluie of lhe lesl in vivo
lesls aie no Iongei avaiIalIe fion SDRU. A suilalIe
'goId slandaid aIleinalive foi lhe in vivo assay is lhe
in vilio Veio ceII assay, vhich is lased on lhe
cyloloxicily of diphlheiia loxin lo cuIluied Veio ceIIs
19
.
FlGURE AIgorithn for the Iaboratory diagnosis of
diphtheria
clinical specimen
screening diagnosis and investigation
ot cases, contacts, and carriers
Tellurite agar, blood agar Tellurite agar (+ blood agar)
18 to 48 b 18 to 48 b
black colonies
Gram positive rods Reference
catalase positive Iaboratory
nitrate pyrazinamidase urease blood agar
4 b 4 b 4 b (pure culture)
Reference Iaboratory
pyrazinamidase cystinase toigenicity tests biotyping
4 b 24 b Llek test: 24 b, 48 b 24 b
L|A: 3 b
PCP: 4 b
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AII lhe nelhods (apail fion lhe nevIy deveIoped
LIA) foi loxigenicily have leen fuIIy docunenled
19,2O
.
Cenolypic nelhods, lased on ICR, offei nany
advanlages ovei slandaid phenolypic lesls: lhey aie
iapid, sinpIe, easy lo inleipiel, and leconing noie
videIy avaiIalIe in Ialoialoiies. These nelhods,
hovevei, do nol shov vhelhei lhe oiganisn can expiess
fuIIy funclionaI diphlheiia loxin. ICR nusl lheiefoie
le used cauliousIy, lecause sone isoIales of C. ipn|ncriac
possess lhe loxin gene lul do nol expiess lioIogicaIIy
aclive loxin
19
. The geogiaphicaI disliilulion of such
sliains is unknovn. Il is lheiefoie advisalIe lo use ICR
onIy as an adjuncl lo a phenolypic lesl, such as lhe LIek.
C. u|ccrans and C. pscuc|uocrcu|csis nay aIso possess
lhe diphlheiia |cx gene and can pioduce diphlheiia loxin,
lul an unequivocaI negalive ICR iesuIl vilh appiopiiale
inleinaI conlioIs viII excIude lhe diagnosis of diphlheiia.
Civen lhe innense pulIic heaIlh inpIicalions
associaled vilh lhe isoIalion of a loxigenic sliain of
C. ipn|ncriac, lhe deIay lelveen isoIalion of a suspicious
oiganisn and lhe iesuIls of loxigenicily lesls can piovoke
gieal anxiely anong Ialoialoiy slaff, cIinicians, pulIic
heaIlh officiaIs, and palienls and lheii faniIies. Il is
inpoilanl lhal aII isoIales of piesunplive C. ipn|ncriac
and C. u|ccrans le exanined foi loxin pioduclion,
lecause of lhe iesuigence of diphlheiia in lhe Laslein
Luiopean Region and lhe endenicily of lhe disease in
nany pails of lhe voiId
21
.
Laboratory responsibiIity for reporting
toigenic C. dphtheroe, C. ulcerons, and
C. pseudotuberculoss
When a piesunplive loxigenic sliain is isoIaled, lhe
foIIoving nusl le infoined innedialeIy: lhe cIinician
iesponsilIe foi lhe case, lhe IocaI consuIlanl in
connunicalIe disease conlioI, SDRU, and lhe IHIS
ConnunicalIe Disease SuiveiIIance Cenlie (CDSC). The
isoIale nusl le senl lo SDRU foi confiinalion.
5usceptibiIity testing of coryneforns
Neilhei lhe iilish Sociely foi AnlinicioliaI
Chenolheiapy (SAC) noi lhe NalionaI Connillee foi
CIinicaI Ialoialoiy Slandaids (NCCIS) of lhe Uniled
Slales (US) has pulIished specific guideIines foi
suscepliliIily lesling of polenliaIIy loxigenic
coiynelacleiia. This nay iefIecl lhe ieIalive iaiily of
lheii isoIalion fion cIinicaI specinens in lhe UK and US.
Il is nol cIeai al piesenl vhich nediun shouId le used
foi gioving lhe cuIluie foi suscepliliIily lesling, noi
vhich nediun shouId le used foi liolh niciodiIulion
oi agai diIulion lechniques. Inleipielive ciileiia shouId
piolalIy iesenlIe lhose ieconnended foi olhei Cian
posilive species such as slieplococci and slaphyIoccci.
The polenliaIIy loxigenic coiynelacleiia aie usuaIIy
susceplilIe lo peniciIIin and nacioIide anlinicioliaIs,
lhese aie lhe agenls ieconnended ly WHO foi
anlinicioliaI liealnenl of cases and caiiieis
5
. AII sliains
isoIaled fion epidenic aieas in easlein Luiope veie
susceplilIe lo eiylhionycin, peniciIIin, anpiciIIin,
cefuioxine, chIoianphenicoI, cipiofIoxacin, genlanicin,
and leliacycIine. Tvo isoIales exhililed ieduced
suscepliliIilies lo iifanpicin, an anliliolic oflen used in
easlein Luiope
22
. In noie iecenl sludies of a gIolaI
coIIeclion of isoIales, aII sliains veie fuIIy sensilive lo
lhe anliliolics exanined (peniciIIin, IevofIoxacin,
ofIoxacin), lul five isoIales fion epidenic aieas in soulh
easl Asia and spoiadic isoIales fion AusliaIia shoved
inleinediale iesislance lo eiylhionycin
23
. These sludies
denonslialed lhe diveise iange of anlinicioliaI agenls
lhal nay le used as aIleinalives lo peniciIIin oi
eiylhionycin shouId lhe need aiise.
5eroIogicaI innunity testing
SeioIogicaI lesling is undeilaken foi seveiaI ieasons,
vhich incIude heIping lo confiin a cIinicaI diagnosis,
lhe assessnenl of individuaI and popuIalion innunily,
and invesligalion of iesponses lo innunisalion in
seIecled individuaIs.
Measuienenl of seiun anlilody IeveIs lo diphlheiia
loxin in a suspecled cIinicaI case, lefoie adninislialion
of anliloxin, nay heIp lo confiin lhe diagnosis,
pailicuIaiIy vhen cuIluies aie negalive. If anlilody
IeveIs aie Iov oi undeleclalIe, lhe diagnosis cannol le
eIininaled. If high, loxigenic C. ipn|ncriac shouId nol
cause syslenic disease. In eilhei case, deleininalion of
anlilody IeveI shouId nol le iegaided as a liuIy
diagnoslic lesl foi diphlheiia and lheiapeulic
inleivenlion(s) shouId nevei le deIayed pending lhe
iesuIls of such lesls.
SeveiaI nelhods foi assessing anlilody IeveIs have
leen desciiled
1
, anong vhich lhe in vivo loxin
neuliaIisalion lesl is iegaided as lhe goId slandaid.
IoilunaleIy, aIleinalive lesls using lissue cuIluie ceIIs
have leen deveIoped as ieIialIe aIleinalives. These
lesls neasuie lhe aliIily of seiun fion an individuaI
lo neuliaIise lhe cylopalhic effecls of puie diphlheiia
loxin in a lissue cuIluie syslen, and lhus neasuie
funclionaI anlilodies. NeuliaIisalion assays aie
pailicuIaiIy usefuI in assessing lhe necessily oi
olheivi se of loosli ng i nnuni ly lhiough
innunisalion. Ciileiia foi inleipielalion of seiun
anliloxin IeveIs aie given in lalIe 6.
LIISA and passive haenaggIulinalion lesls have aIso
leen deveIoped and used foi neasuiing seiun anliloxin
IeveIs. The alliaclion of lolh lhese nelhods is lheii speed
and convenience, lul seveiaI iepoils have suggesled lhal
lhey nay le Iess ieIialIe lhan lissue cuIluie
neuliaIisalion assays. Iassive haenaggIulinalion assays
have leen found lo Iack sensilivily foi seiun specinens
conlaining <O.1 IU/nI anliloxin, vilh an ensuing iisk
of faIse negalive inleipielalions. SiniIaiIy, LIISA lesls
have leen shovn lo coiieIale pooiIy vilh lissue cuIluie
and in vivo neuliaIisalion lesls foi specinens conlaining
<O.1 IU/nI anliloxin, and lheiefoie caiiy a significanl
iisk of giving faIse posilive iesuIls. The LIISA faIse
posilive ieaclions aie leIieved lo le due lo lhe linding
of non-neuliaIising anlilodies. LIISA lesls have leen
used foi pieIininaiy scieening of seiun specinens, lhese
aie ie-lesled ly lissue cuIluie/in vivo neuliaIisalion
assays if lheii anliloxin lilies aie <O.1 IU/nI. Oui
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Ialoialoiy has nol adopled lhis appioach lecause il has
leen iepoiled lhal LIISA nay nol le a ieIialIe piedicloi
of innunily even foi specinens vilh anliloxin
concenlialions > O.1 IU/nI.
5peciaIised testing, noIecuIar typing
SeveiaI nel hods have leen deveI oped and
evaI ual ed ly SDRU and l he nenleis of l he
Luiopean Ialoialoiy Woiking Cioup on Diphlheiia
( LIWCD) l o i nvesl i gal e l he noI ecuI ai
epi deni oI ogy of C. ipn|ncriac. These incIude
iilolyping, puIsed fieId geI eIecliophoiesis, ICR
lypi ng, si ngI e slianded confi inali onaI
poIynoiphisns (SSCI) of lhe |cx gene, and noie
iecenlIy anpIified fiagnenl Ienglh poIynoiphisns
(AIII)
24-27
. The nosl lhoioughIy evaIualed and
di scii ni naloiy nelhod lhus fai appeais lo le
iilolyping and dalalases as desciiled leIov have
leen eslalIished using lhis lechnique
28
.
The 'DipIdenl dalalase of noIecuIai lyping
palleins of C. ipn|ncriac has leen eslalIished anong
nenleis of LIWCD. Ils cenliaI dalalase is al lhe
Inslilul Iasleui in Iaiis and a sul-dalalase ('DipRilo
UK) is heId al lhe SDRU. The dalalase conlains
dala fion lhe exaninalion of ovei 6OO isoIales of
C. ipn|ncriac fion epidenic and non-epidenic aieas
voiIdvide, using lhe slandaidised iilolyping schene
agieed ly LIWCD. In liief, lhis consisls of a iilolype
piolocoI, vhich incIudes DNA iesliiclion vilh Ptu||
and 8s|||| and hyliidisalion vilh a nixluie of five
digoxigenin-IaleIIed oIigonucIeolides laigeling
univeisaIIy conseived iegions of 16S and 23S iRNA.
The palleins aie scanned using a 256-giey scannei and
inleipieled using lhe Taxc|rcn piogianne
28
. Thus
fai, noie lhan 7O diffeienl palleins have leen
idenlified, vilh lhe piedoninanl palleins fion lhe
Laslein Luiopean Region leing piovisionaIIy designaled
as 'D1 and 'D4 (D iepiesenling diphlheiia, lased upon
piovisionaI nonencIaluie of lhese genolypes). The
nonencIaluie is cuiienlIy leing finaIised ly LIWCD.
This dalalase aIIovs us lo idenlify lhe nosl IikeIy
geogiaphicaI oiigins of pailicuIai isoIales and lheii
ieIalionship lo endenic and epidenic sliains voiIdvide.
Iion lhis ve have confiined lhal heleiogeneily lelveen
sliains fion diffeienl iegions exisls and lhal dalalase
sloiage aIIovs lhe iecognilion, confiinalion, and
idenlificalion of appaienl cIones voiIdvide.
CIinicaI appIication
These nelhods veie used in lhe invesligalion of lhiee
cases of loxigenic C. ipn|ncriac infeclion lhal aiose in
lhe UK in 1997.
The fiisl case vas a 4O yeai oId nan vilh culaneous
infeclion vilh loxigenic C. ipn|ncriac vai ni|is vho
had iecenlIy ieluined fion Indonesia. He had leen
fuIIy innunised in chiIdhood and had ieceived a
looslei lefoie liaveIIing in 1996. One asynplonalic
househoId conlacl vas found lo have a posilive lhioal
cuIluie. Neilhei lhe case noi lhe conlacl had signs of
loxicily noi appaienl lhioal infeclion. IsoIales fion
lhe index case and lhe conlacl veie genolypicaIIy
indislinguishalIe ly iilolyping and iesenlIed
pievious isoIales fion Indonesia.
In lhe second incidenl an uninnunised vonan aged
72 yeais deveIoped a soie lhioal duiing a aIlic ciuise.
The IocaI Ialoialoiy Iiaised vilh SDRU and, lo ieduce
any polenliaI deIay in confiining lhe cIinicaI diagnosis,
sanpIes of lhe nenliane veie senl diieclIy lo lhe unil.
A loxigenic sliain of C. ipn|ncriac vai gratis vas isoIaled
fion lhe nenliane vilhin 24 houis of ieceipl. Iuilhei
chaiacleiisalion of lhe isoIale ly iilolyping shoved lhal
lhe pallein pioduced vas indislinguishalIe fion lhe
'epidenic pallein D1 lhen ciicuIaling in easlein Luiope.
This vas lhe fiisl inpoilalion of D1 lo lhe UK fion lhe
Laslein Luiopean Region
16
.
The lhiid incidenl vas a C. ipn|ncriac infeclion
acquiied al voik ly a hospilaI Ialoialoiy voikei vho
had handIed a loxigenic oiganisn in a sanpIe
disliiluled ly lhe NalionaI LxleinaI QuaIily Assuiance
Schene (NLQAS) foi niciolioIogy in Novenlei 1997.
Infoinalion suppIied vilh lhe sanpIe said lhal il cane
fion a palienl vilh seveie soie lhioal vho had iecenlIy
ieluined fion Russia. The cIinicaI isoIale, lhe isoIale
used in lhe NLQAS sanpIe, and lhe oiiginaI souice
cuIluie piovided ly SDRU lo NLQAS veie
indislinguishalIe ly iilolyping and veie found lo
leIong lo iilolype 'D2O, an unconnon pallein, seen
onIy once lefoie, in a palienl fion Kazakhslan
1O
.
Enhancing nicrobioIogicaI surveiIIance
Iuilhei inpiovenenls lo pioficiency in Ialoialoiy
diagnosis aie leing addiessed in lhe IHIS ly lhe
oiganisalion of speciaIised voikshops on lhe diagnosis
of diphlheiia foi IHIS and NHS Ialoialoiy peisonneI.
Such voikshops have leen heId once oi lvice yeaiIy
foi UK voikeis since 1994. Woikshops heId in
conjunclion vilh WHO foi peisonneI fion counliies in
Luiope (as pail of lhe Luiopean Connission funded
piogianne on lhe 'MiciolioIogicaI SuiveiIIance of
Diphlheiia in Luiope) have aIso laken pIace. Thiough
such voikshops, lhe WHO ManuaIs, lhe IHIS SOIs,
iepoils in CDR lcc||q, and ieguIai Iiaison vilh
inleinalionaI agencies (foi exanpIe, WHO) lolh cIinicaI
and niciolioIogicaI avaieness has inpioved in lhe pasl
fev yeais. Ialoialoiy skiIIs in lhe iecognilion of lhese
palhogens shouId nol le aIIoved lo decIine as lhe disease
is fai fion eiadicaled voiIdvide
29,3O
.
TABLE 6 lnterpretation of neasured antitoin IeveIs as
defined by the in vitro neutraIisation assay
Antitoin level
(|U/mL) |nterpretation
<0.01 |ndividual is susceptible
0.01 Lowest level ot circulating antitoin giving
some degree ot protection
0.01 - 0.09 Levels ot antitoin giving some degree ot
protection
0.1 A protective level ot circulating antitoin
>1.0 A level ot antitoin giving long term
protection
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AcknowIedgenents
We lhank lhe slaff of SDRU foi lheii dedicalion and haid
voik, in pailicuIai, lhose undeilaking lhe 'oul of houis
seivice - Cina Mann, Iiances Knighl, Siolhan Mailin,
Daksha Halhi, Tony Mc Niff, Nila Doshi - and aIso Kalhy
LngIei and Aiuni De Zoysa. We aIso lhank oui
coIIeagues in lhe Innunisalion Division al CDSC, in
pailicuIai Noinan egg, }oanne While, and Maiy
Ransay. Oui lhanks lo coIIeagues in lhe IHIS and NHS
Ialoialoiies foi lhe sulnission of isoIales, infoinalion,
and foi lheii coIIaloialion.
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