Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples

0095-1137/94/$04.00+0

Copyright X)1994,AmericanSociety forMicrobiology

Nested PCR Optimized

for Detection of

Bordetella

pertussis

in

Clinical

Nasopharyngeal

Samples

ANDERS BACKMAN,1 BO JOHANSSON,2ANDPER OLCENl*

Departmentof ClinicalMicrobiology andImmunology, Orebro Medical CenterHospital, S-70185 Orebro,' and The Central

Microbiological

Laboratory of

the Stockholm

County

Council,

S-107 26

Stockholm,2

Sweden

Received 29April 1994/Returned for modification 8 June1994/Accepted 11 July 1994

Several genes and sequences in Bordetellapertussis have been usedas targets indiagnosticPCRassays.A previously developed single-stepPCRassayfor thedetection of B.pertussiswasbasedonaninsertion sequence, IS480, that is present in about 70 to 80 copies in each genome. The diagnostic sensitivity, specificity, and

reliability of thisassaywith aspiratedand heat-treatedsamplesfrom thenasopharynxofpatients andtheir contactswasimproved bytheuseofa nestedPCRconfiguration.The nested PCRassayproduceda 205-bp fragmentwith all of the 115 B.pertussis strains tested andwas negativewith all strains belonging to other Bordetella species (n = 44)aswellasother bacteriacommonlyfoundin theupperrespiratorytract(n =115).

The diagnostic value of the assay was verified by giving positive results for B. pertussis in all the 51 nasopharyngeal aspiratesfromculture-positive patients.Theassayalso detected 18positive aspiratesfroma

totalof196 culture-negative patients.Aconfirmatory cleavageof the205-bpnested PCRproductbyMvaIgave

inallcasestwobands of88 and 117bp.Inconclusion, thisnewly developednestedPCRassaywas shownto be reasonably fast and uncomplicated, with an optimal sensitivity and a high degree ofspecificity for the diagnosis of B. pertussis in aspirated nasopharyngeal samples processed simply by heat treatment. The detection level in the nested PCRwasabout 10 bacteriaperml,or seventoeightinsertionsequencecopiesper

10 ,Ilof boiledsample.

The specific diagnosis ofpertussis is important for several

reasons, e.g., fortreatment andepidemiological interventions

as well as evaluation of vaccine efficiency. To date this has mainly depended on culture andserology. Culture has,

how-ever, a low sensitivity (5) and serology is not helpful in the acutephase of thedisease,and the culture result for samples from, for example, vaccinated children can be difficult to

interpret (7). Antigen detection by immunofluorescence and enzyme-linked immunosorbent assay techniques as well as

testsforadenylate cyclase activity (2)have therefore been tried

asdiagnostictests. Inaddition,DNAhybridization (3, 19, 22) andPCR(24)assayshavebeendevelopedinrecentyears(4). The PCR method is sensitive and reasonably fast. Several different regions in Bordetella pertussis DNA, such as the

repeatedinsertionsequence(4, 9, 11, 13, 18, 26),theadenylate cyclasetoxin gene (1), the DNA upstream of theporin gene

(16), the DNA in the 16S-23S rRNA spacer sequences (23), andthepertussistoxinpromoterregion(6, 11, 21), have been used astargetsforthismethod.

Apreviousstudy (18)wasbasedon asingle-step PCR for the

diagnosisofpertussisbydetection ofa400-bp fragment of the

insertionsequenceIS480 (12, 14, 15),which is about 1,050 bp

long and existsat70to80 copiespergenomeinB. pertussis(4, 9, 13, 26).Nocross-reactionswere seenwiththe other species

withinthegenusBordetella.However,IS480has been shownto

hybridize to some extent to single strains of Bordetella bron-chiseptica (4),aspeciesnotknowntocausedisease in humans.

This hybridization probablyoccurs inaregion outside of the

region amplified in the PCR. A problem with the use of the

single-step PCR technique forroutine clinical diagnosis isthat it sometimes fails to amplify B. pertussis DNA in complex

*Corresponding author. Mailing address: Department of Clinical

Microbiology and Immunology, OrebroMedical Center Hospital, S-701

85Orebro, Sweden. Phone: 46-19-151520.Fax: 46-19-127416.Electronic

mailaddress:ulf.e@se.orebroll.adb.

clinical nasopharyngeal samples because of the presence of inhibitors (25) or other factors. In a nested PCR assay (17) DNA is first amplified with a set of outer primers. This is followed by a second amplification with so-called nested primers covering a selected sequence within the primarily amplified DNA sequence. An advantage with a nested PCR system is that it gives a significant dilution of the original material, including inhibitingsubstances. In addition, theuse

ofanested systemtheoretically givesabuilt-in confirmationof theprimarily amplified region bythe nestedprimers.Inorder to increase the efficiency of the single-step PCR assay (18), which hadasensitivityof 0.76 inaprevious study (18),anested

pair of primers was designed with the aim of obtaining a

robust, sensitive,andspecific PCRassayfor themoreefficient

diagnosis of B.pertussisin nasopharyngeal samples.

MATERIALSANDMETHODS

Patients andsampling. Samples from consecutivechildren hospitalized or attending the outpatient clinic of the Depart-mentofPediatricsof theOrebroMedical Center Hospital with clinical symptoms ofpertussis (n = 75) were obtained from

JanuarytoDecember 1990. In addition, samples from

consec-utive children and theircontactswith clinical symptoms

sug-gesting pertussis and participating in a national vaccination

study (n= 172)wereobtained from January1992toDecember 1993. ForthePCRassaynasopharyngealsamplesweretaken

by aspiration with a prefabricated kit for tracheal suction

(Intermed; Nunc, Roskilde, Denmark). After suctionviaone

of the nostrils for 3 s the catheter was withdrawn. In the

samplesfrom1990, 5to10ml of physiological salinewasthen

aspirated to wash the catheter and the collected fluid was

frozenat-70°C within3to4h. In the samples from1992 and 1993, 1mlofphosphate-buffered salinewasusedtowash the catheter.Thetipofthecatheterwasalso asepticallyputinthe enrichmentmediumofRegan andLowe (20)for culture.The 2544

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NESTED PCR FOR DETECTION OF B. PERTUSSIS IN ASPIRATES 2545 TABLE 1. Bacterial strains used in the nested PCR assay

for B.pertussis DNAa

Bacteria _isolatesNo. of

Bordetella

pertussl'

... 115

Boraetella oroncniseptica... Bordetella avium... Bordetella species... Alcaligenes species... Haemophilus influenzae... Haemophilusparainfluenzae... Moraxellacatarrhalis... OtherMoraxella species... Neisseriameningitidis...

Neisseria gonorrhoeae... Other Neisseria species... Streptococcus pneumoniae... Streptococcus groups A, B, C, and G ... Alphastreptococci... Staphylococcusaureus... Staphylococcusepidermidis... Diphtheroid rods... Bacteroides species... Anaerobicstreptococci... Fusobacteriumspecies... Propionibacterium acne... 32 7 3 2 5 22 4 4 4 7 4 10 12 14 7 4 4 4

aThetotal number ofstrainstestedwas274.

bAll115 isolates hadapositive PCR result. All the other bacterial strains in

thetable hadanegative PCR result.

sampleswerekeptat -70°C andwereneverthawedmorethan

twicebeforebeing assayed.

Culture. In 1990 thenasopharyngealsamples for culture for B.pertussisweretakenviaonenostril withadacronswab(ENT

Swab) thatwasimmediatelyplaced in the enrichment medium

before transport within 3 h to the Department of Clinical Microbiology and Immunology. The swabswere cultured on

enrichedmedium (charcoalagarwith10% defibrinated horse blood and40mg ofcephalexin perliter; Oxoid, Basingstoke,

UnitedKingdom)at36°Cwithhigh humidity, and the cultures

wereexamined for growth of B.penussis-likecolonies forupto 7 days. The cultured swab was reinserted in the transport medium immediately after culture andwasrecultured in the samewayafter 3days of incubationat36°C. In 1992 and 1993, culture forB.pertussiswas performedon theaspirate (swabs were not taken) in the same wayby inoculating three drops

(approximately90,ul)ofaspirated fluid fromaPasteurpipette.

Thetip of thechoppedcatheterwasalsocultured after 3 days

ofincubationat36°C.

The diagnosis of B. pertussis was based on typical colony

forms, direct microscopic observation of short gram-negative

rods, oxidase and catalase positivity, a specific reaction with

agglutinatinganti-B.pertussisserum(producedatthe National Bacteriological Department, Stockholm, Sweden, or

pur-chasedfrom DifcoLaboratories), and anegative reaction by

the agglutination testwith anti-B. parapertussis serum (Difco

Laboratories).

Bacterial strains. In order to examine the sensitivity and specificityof thenestedPCRassay266 clinical and 8 reference strainswereused(Table 1).Theclinical strainswerechosento representthegenusBordetella and bacteriacommonlyfound in the upper respiratory tract. These strains were collected be-tween 1983 and 1991 (Bordetella species) and between 1976 and 1991 (other bacteria)fromOrebro County,Sweden. The AmericanType Culture Collection (ATCC)reference strains included were B. pertussis ATCC 9340, ATCC 9797, and

TABLE 2. Results by nested PCR for B.pertussis in relation to results by nasopharyngeal culture of swabs andaspiratesa

No.of samples with the indicated culture result: Nested PCR Swab Aspirate

result Swab

Positive Negative Total Positive Negative Total

Positive 25 7 32 26 11 37

Negative 0 43 43 0 135 135

Total 25 50 75 26 146 172

aSwabswereobtained in1990, and aspirateswereobtained in1992and1993.

ATCC 9340, B.

parapernussis

ATCC 15311 and ATCC 15237, B. bronchiseptica ATCC 786 and ATCC 19395, and B. avium ATCC35086.

Nested PCR assay and restriction enzyme verification of amplifiedproduct. AnestedPCRmethodwasdevelopedfrom the single-step PCR assay described before (18). The new

primerswere designed to give a 205-bpfragment (base pairs

634to839onIS480) (12). ThePCR assays wereperformedin alaboratorywhere the handling of reagents and samples was

physicallyseparate from that ofamplifiedmaterial. As positive and negative controls, a clinical isolate of B.pertussis (strain 91-12-0222; 7 x 104/ml in water) and a nasopharyngeal aspi-ratefrom ahealthy individualwere used,respectively.Controls were included ineach amplification, andthe clinical samples wereassayedandinterpretedwithout knowledge of the culture

results. Thenasopharyngeal sampleswere boiled for 30 min in

cappedEppendorf tubes, and 10 ,ul was added togivea final

PCR mixtureof 30

,u1

containing lx reaction buffer (1.5mM

Mg2";

Perkin-Elmer

Cetus),200

p.M

deoxynucleoside

triphos-phates (Perkin-Elmer Cetus), 1 U of AmpliTaq polymerase (Perkin-Elmer Cetus), 15% glycerol, and 0.33

p.M

(each) primer

(5'-GAClTTCGTCTFlCGTGGCCAT-3'

and 5'-GTA

CAGCGCGCCCGATGCCT-3'; Symbicom,

Ume'a,

Sweden).

The reaction mixture wascoveredwith mineral oil and ampli-fiedin a DNAthermalcycler(Perkin-ElmerCetus).Eachcycle consisted of temperaturesteps at94, 50, and 72°Casdescribed before (18). The first round of the nested PCR was for 20

cycles.Fromthisreaction mixture a

1-pI

samplewasadded to a newPCR mixture(29

,ul)

containingthe newnestedprimers

(5'-CGCGTGGCCTTCACCGACAT-3'

and 5'-GGGCGGT

AAGGTCGGGTAAA-3'; Scandinavian Gene Synthesis AB,

Koping,

Sweden). This PCR was rununder the same

condi-tions describedabove,butfor30cycles.The PCRproduct (8

p.l)

was assayed on a 3% agarose gel (Nusieve GTG; FMC

Bioproducts) with 40mM TAE(Tris-acetate, EDTA) buffer

andethidiumbromide and the bandpositionswerecompared

with thoseofDNAmolecularweight marker V(Boehringer, Mannheim, Germany) as described before (18). To confirm

theidentity oftheproduct, allculture-negative samplesanda randomselection of theculture-positive samplesweredigested

with MvaI for 1 h at37C andwereelectrophoresedon a3.5%

agarosegel.Thedetectionlimit wasinvestigated by

amplifica-tion of boiled dilutions of B. pertussis 91-12-0222 in the

nasopharyngeal

aspirate fromahealthy individual.Theresults

were analyzedon anagarose gel asdescribed above.

RESULTS

Thenested PCR assay gave a

205-bp

product

with all 112

clinical isolates of B.pertussis inwater

(7

x 104 bacteriaper

ml),the three ATCC strains ofB.pertussis,andamixtureof B. pertussis, Haemophilus

influenzae,

Streptococcus

pneumoniae,

and aMoraxella

species

(each

with 106 bacteria per

ml).

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1234567891011

FIG. 1. Agarosegel electrophoresisafter nestedPCR ofB.

pertus-sis DNA in nasopharyngealsamples. Lane 2, PCR-positiveresult for

swab culture-positive sample; lane 3, PCR-positive result for swab

culture negative sample; lane 4, PCR-positive result for aspirate

culture-positive sample; lane 5, PCR-positive result for aspirate

cul-ture-negative sample. Thebands (205 bp)werecompared with the

192-and 213-bp b192-ands (markedwith arrows) of themolecularmassmarker

in lane 1.

otherBordetella species (n = 44) and other respiratory tract

strains (n = 115) were negative in the assay (Table 1). The nested PCRwaspositivewith all 51 nasopharyngealaspirates from B. pertussisculture-positivepatients (Table 2 andFig.1). No strains of B.parapertussisorB.bronchisepticawereisolated

from this material. A random selection of 14 of these 51 positive PCR productswassubjectedtoMvaIcleavage. All of themgavetwobands of the expected sizes forB. pertussis, i.e., 117 and 88 bp (Fig. 2). The nasopharyngeal aspirates from culture-negative patients (n = 196)were negative by nested

PCR for 178 of thesamples and positive for 18 of the samples

1

714_

FIG. 2. Agarose gel electrophoresis after nested PCR and MvaI

cleavageof B.pertussisDNA in thenasopharyngeal samples presented

inFig.1. Lane2, positiveresult for swabculture-positive sample;lane

3, positive result for swab culture-negative sample; lane 4, positive

result foraspirate culture-positive sample; lane5, positive result for

aspirate culture-negative sample. The bands (117 and 88 bp) were

comparedwiththe 89- and123-bpbands(markedwitharrows)of the

molecularmassmarkerinlane1.

FIG. 3. Agarose gelelectrophoresis afternested PCR of dilutions

ofheat-treatedB. pertussis 91-12-0222 in a nasopharyngeal aspirate

fromahealthy individual.Lane 2, originalsuspension; lanes 3to9,a

series ofdilutions with 100, 50, 25, 10, 5,1, and0.5 bacteriaperml,

respectively; lane10, anegative control ofanasopharyngeal aspirate

fromahealthy individual; lane11,positive control. The bands(205 bp)

werecompared with the 192-and 213-bp bands of the molecularmass

marker in lane 1. Afaint but clearband could beseen onthe original

gelinlane 6.

(Table 2; Fig. 1, lanes 3 and 5).All 18 PCR products could be verified by MvaI cleavage (Fig. 2, lanes 3 and 5). Altogether, the sensitivity ofthe nested PCR assay for the diagnosis of

infection caused by B.pertussis in thetwo clinicalgroups was

1.00. Thespecificitywas0.86 whenswab culture and 0.92 when

aspirate culturewereusedasthe reference methods (Table 2). The detectionlevelfor B. pertussis inanasopharyngeal aspirate

from a healthy individual by the nested PCR assay was

determined by serial diluting boiled material to about 10B. pertussis cellsperml(Fig. 3), correspondingtoseven toeight copiesof thetarget sequence persamplefor the nested PCR.

DISCUSSION

The nested PCR assay for the diagnosis of B.pertussis in nasopharyngeal aspirates showed an optimal sensitivity in clinicalsamples.Thetargeted regionwasalso documented in all 115 strains of B. pertussis examined. With culture as the referencemethod fordiagnosis,somesampleswereidentified

to be culture negativeand nested PCRpositive. Thisfinding

was expected because of the well-known fact that culture is falsely negative in a proportion of cases (5). This is mainly dependent onwhen in thecourse ofthe disease the samples

were obtained from the patients, the sampling technique,

meansand times oftransport (10),culturesystemandmedium, and age and whether the patient had been vaccinated or

treated with antibiotics. Samples for culture were obtained

fromourpatientsearlier in the course of disease during the

1992 to 1993 period (data not shown), which may be one

explanationofwhy only8%wereculturenegativeand nested

PCRpositive duringthisperiodwhereas 14% from the

previ-ous period were culture negative and nested PCR positive. Anotherfactor could be that culture ofnasopharyngeal

aspi-rateis moreefficientthan culture ofswab,as documentedby Hallanderetal.(7).

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NESTED PCR FOR DETECTION OF B. PERTUSSIS IN ASPIRATES 2547 For further exploration of the specificity of the nestedPCR,

a"gold standard" for the diagnosis must be created by using not only culture but also serology and preferably also PCR assays covering other parts of the genome. A study that will use

this approach is planned. In clinical samples the presence of

inhibitorsof the PCR assay is always a potential problem, and

different approaches, depending on the clinical material, to overcoming thesedifficultiesmust be evaluated. For the frozen nasopharyngeal samples, boiling in combination with the

dilu-tion which takes place during the nested PCR seems to overcomethis problem in the diagnosis of pertussis by PCR. It would, however, be helpful to have an indicator of the perfor-mance of the PCR assay and a possibility of obtaining a quantitative measure of the original amount of B. pertussis DNA. By addingdefinedamountsofa labelled piece of IS480 to every clinical sample, a competitive assay which can give

informationabout theinhibitoryactivities in thesampleaswell as a quantitative numeric measure can be designed; this

approach is being evaluated. The role offreezing compared

with that of examination of fresh samples should also be evaluated. In some situations the diagnosis of B.

parapertussis

isimportant,althoughtheimpactonhumanmorbidityismuch

less, and epidemics hardly exist. In vaccination studies a

diagnostic PCR assay for B. pertussis

ought

to be

included,

whilediagnosis ofB.

bronchiseptica

infectioncouldbe

consid-ered less important unless it creates

problems

with

false-positive results intestsfor the otherBordetella

species

(8).

Recommendationsfor theuseofPCR for the

diagnosis

of B.

pertussishave been

proposed

(16),

and the assaydescribedhere

complieswith most of those

guidelines.

The assayworks for

both

aspirate

and swab

samples,

and no extraction step is

necessary, thus

making

the test

simple

andreliable.The assay

was

designed

to detect B.

pertussis

and no other Bordetella

species.The

sensitivity

wasvalidated

by positive

results forall B.pertussis

culture-positive

samples

and all B.

pertussis

strains

obtained from different

periods

between 1983 and 1991.

Strains isolated from other

geographic

areas remain to be

examined.A

false-positive

controlwasmaintained

by

physical

separation

of the reagent mixture

preparation,

sample

storage

and pretreatment,

amplification,

and

gel electrophoresis

with

subsequent

photography.

The results for the coded

samples

with

positive

results were confirmed with

typical

restriction

enzyme

fragments

on a

gel.

A

problem

with the nested PCR assay

compared

with a

single-step

PCR assay as a routine

diagnostic

method can be the increased risk of carryover of

amplified

DNAand thefactthat thiscannotbe combatedwith

the

uracil-N-glycosylase

carryover

prevention

system.

There-fore,

we recommend that

only

laboratories with

thorough

experience

with PCR use the nested PCR

technique

for

routine

diagnostic

purposes.

In conclusion the set of

primers

and the nested PCR

approach

usedin thepresent

study

seemtobe

powerful

tools

anda

complement

toculture in the

diagnosis

of

pertussis

in the

acute stage of disease. This

improvement

in the

diagnosis

of pertussis would

substantially

strengthen

the assessment of

clinical

vaccine trials.

ACKNOWLEDGMENTS

Thisstudywas

supported by

grantsfrom the OrebroCountyCouncil Research Committee and the Foundations for Medical Research,

Orebro

Medical Center

Hospital.

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