J Clin Pathol 1993;46:313-317

Detection of Chlamydia pneumoniae andChlamydia psittaci in sputum samples by PCR

C YW Tong, M Sillis

AbstractAims-To use the polymerase chainreaction (PCR) to detect Chlamydiapneumoniae and Chlamydia psittaci insputum samples.Methods-A nested PCR was developed,the first stage of which amplified DNAfrom both C pneumoniae and C psittaciwhile the second stage targeted specifi-cally at C pneumoniae, allowing the twospecies to be differentiated. The primerswere designed not to amplify sequencesfrom C trachomatis. A panel of 26 spu-tum samples from patients with commu-nity acquired pneumonia evaluatedpreviously by enzyme linked immuno-sorbent assay (ELISA), direct immuno-fluorescence (DIF), and culture wastested blind by PCR. Most of thesespecimens also had accompanying serialserum samples which were tested forspecies specific antibodies using micro-immunofluorescence (micro-IF).Results-PCR detected C pneumoniaeDNA in 10 of the 26 samples and Cpsittaci DNA in four. There was goodconcordance between ELISA, DIF,micro-IF and PCR in the C pneumoniaegroup. Two of the C psittaci identified byPCR were labelled Cpneumoniae by DIFbut the PCR results were supported byserology or a history of bird contact. Ofthe PCR negative group: six were truenegative results; two contained C tra-chomatis. There were four discrepantresults.Conclusions-The data suggest that PCRis effective in the detection of Cpneumo-niae. The sensitivity for C psittaci isinevitably lower due to the strategy takenbut specificity seemed to be good.

(7 Clin Pathol 1993;46:313-317)

Chlamydia pneumoniae and Chlamydia psittaciare important causes of lower respiratory tractinfections. The reported prevalence of C

Table 1 Sequence and position of the primers on the ompA gene of C pneumoniae

Primers Sequence Positions

External 333 base pair productsCPl (sense) 5' TTA CAA GCC TTG CCT GTA GG 3' 61-80CP2(anti-sense) 5' GCG ATC CCA AAT GTT TAA GGC 3' 373-393

Internal 207 base pair productsCPC(sense) 5' TTA TTAAT GAT GGT ACA ATA 3' 100-120CPD(anti-sense) 5' ATC TAC GGC AGT AGT ATA GTT 3' 286-306

pneumoniae in community acquired pneumo-nia has ranged between 10-20%.'-3 C psittacihas remained an important pathogen in theUnited Kingdom and has been associatedwith the import of psittacine birds4 and withdomestic poultry.5 Despite the importance ofthese two organisms, early and reliable meth-ods for laboratory diagnosis are not available.Isolation from clinical specimens is difficultfor C pneumoniae6 and hazardous for Cpsittaci.7 Though well established for C tra-chomatis,8 9 the value of enzyme linkedimmunosorbent assay (ELISA) and directimmunofluorescence (DIF) for detectingthese two organisms in respiratory sampleshas had only a preliminary evaluation.'011Due to the heterogeneity of C psittaci as agroup, no species specific reagent is available,making species differentiation difficult.Serology, particularly microimmunofluores-cence (micro-IF), has been the mainstay ofdiagnosis, but the retrospective nature of thistest, due to the late appearance of speciesspecific antibodies, has made it unhelpful inclinical management.

Several groups have explored the possibil-ity of detecting C pneumoniae or C psittaciusing the polymerase chain reaction(PCR).'4 However, these reports providedvery little information on the use of PCR onclinical specimens, particularly on sputumsamples from patients with pneumonia. Amethod to detect and differentiate C pneumo-niae and C psittaci in sputum samples usingPCR has been developed.

MethodsThe major outer membrane protein genes(ompA) of C pneumoniae and C psittaci werechosen as the target for amplification in anested PCR. All primers were synthesisedusing an oligonucleotide synthesiser (AppliedBiosystems). The outer primers (table 1)amplified a 333 base pair product from bothorganisms. The primers were chosen so asnot to amplify the ompA gene of C trachoma-tis. One of the internal primers, designatedCPD, was located in a variable domain of theompA gene and was specific for Cpneumoniae."' 16 Hence only the first stageproduct from C pneumoniae was exponentiallyamplified to yield the 207 base pair secondstage product, allowing the two organisms tobe differentiated.

Sputum samples were prepared and con-centrated as described before.'7 Before PCR,the concentrated specimens were diluted ten-

Department ofVirology, RoyalVictoria Infirmary,Newcastle Upon TyneC YW TongPublic HealthLaboratory,Bowthorpe Road,NorwichM SillisCorrespondence to:Dr C Y W TongDepartment of MedicalMicrobiology University ofLiverpool Duncan Building,Royal Liverpool HospitalLiverpool L7 8XPAccepted for publication24 September 1992

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fold in PCR buffer (10 mM TRIS-HCI, pH8&3, 50 mM KCl, 1-5 mM MgCl,, 0O1%Triton X-1 00) and digested with proteinaseK (60 ug/ml) at 55°C for 60 minutes. Afterdigestion, proteinase K was inactivated byheating to 1 00°C for 10 minutes and thesamples were cooled on ice. Ten microlitresof the treated samples were subjected to PCRunder mineral oil overlay in the same PCRbuffer with 044,M each of CP1 and CP2primers (table 1), 200,iM each of dATP,dTTP, dCTP and dGTP (Pharmacia) and2-5 units of Taq DNA polymerase(Northumbria biological laboratories).Amplification was done with a thermocycler(Perkin-Elmer) using the technique of"Touchdown" PCR'8 in which the annealingtemperature was lowered 1°C every twocycles from 65°C until touching down to55°C at which temperature 20 more cycleswere performed. The denaturation tempera-ture and the extension temperature were con-stant at 94°C and 72°C, respectively. Theholding time at each temperature was 1minute.The PCR products amplified by the outer

Agarose gel electrophoresis ofPCR products-first stage in upper part and second stage inlower part of the gel. Lanes 1-3: tissue culture fluid (1 C trachomatis serovarA strainSA 1; 2 C psittaci strain 33L-human; 3 C pneumoniae strain IOL 207). Lanes 4-7:sputum samples (4 case 18 - negative; 5 case 13-C psittaci; 6 case 2-C pneumoniae,low positive; 7 case 4-C pneumoniae, strong positive). Lane 8pGEM molecular sizemarker (Promega).

primers were diluted 10 times in water, and10 lul was transferred to a PCR reaction mixfor amplification in a second stage using theinner primers. The PCR reaction mix was thesame as before except that 1 puM each of theprimers CPC and CPD (table 1) were usedand the MgCl, concentration was increasedto 3 mM. The second stage PCR consisted of30 cycles of 94°C, 50°C, 720C each for 1minute. Both the first and second stage prod-ucts were analysed in 3% agarose gel elec-trophoresis (Seakem LE:Nusieve GTG 1:3,FMC Bioproducts) with ethidium bromidestaining by standard techniques.'9 PCR wasperformed in the Virology Department ofRoyal Victoria Infirmary, Newcastle UponTyne which did not provide routine chlamy-dial diagnostic service, and no clonedchlamydial genetic materials were handled inthe laboratory. All samples were testedblindly and repeated at least twice beforereporting. Throughout the procedures, stan-dard precautions were adopted to avoidcross-contaminations.20The above methods were initially evaluated

and optimised using tissue culture materialsfrom the Institute of Ophthalmology,London. All the serovars of C trachomatis, 10strains of C psittaci (three human, four feline,one avian, one ovine, one guinea pig) and twostrains of C pneumoniae (IOL 207 and TW183) were tested. Twelve species of bacteria(Streptococcus pneumoniae, Haemophilusinfluenzae, Staphylococcus aureus, Neisserialactamica, Escherichia coli, Klebsiella oxytoca,Proteus mirabilis, Pseudomonas aeruginosa,Salmonella typhimurium, Streptococcus pyo-genes, Enterococcus faecalis, Mycoplasma pneu-moniae) were included to rule out possiblecross-reactions. The sensitivity of the test wasassessed by comparing PCR results with ele-mentary body counts based on DIF.Twenty six sputum samples from patients

with community acquired pneumonia, exam-ined initially for evidence of chlamydial infec-tions using ELISA (IDEIAIII, DakoDiagnostics), DIF, and cell culture, wereselected for testing using the described PCRmethod. Most of these samples had accom-panying serial serum samples which weretested by micro-IF in the Institute ofOphthalmology, London.DIF was performed on all samples using a

genus specific monoclonal antibody (Imagen,Dako Diagnostics). Presence of three or moreEBs with the characteristic morphology, size,and fluorescence was regarded as a positiveresult. Some of the samples were alsoanalysed with species specific monoclonalantibodies (C trachomatis-Syva, C pneumo-niae Cellabs). Specimens positive for genusspecific antigen but negative for species spe-cific antigen were interpreted as C psittaci.Cell culture was performed using McCoycells as described'7 and stained with the abovemonoclonal antibodies. Micro-IF was per-formed as described.2' A single titre greaterthan 1/256, positive IgM or a fourfold shift intitre were taken as diagnostic of recent infec-tion. A stable titre of 1/256 in a patient with

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Detection of C pneumoniae and C psittaci in sputum samples

Table 2 Combined results of all the tests done on the panel of 26 specimens

DIFCase BirdNo contact ELISA Genus Species Culture Micro-IF PCR

1 No + + C pneumoniae - Cpneumoniae 1:256 C pneumoniae2 NK + + + ND - Cpneumoniae 1:256-K 1:64 Cpneumoniae3 Yes + + + + C pneumoniae C pneumoniael C pneumoniae 1:512 C pneumoniae

C psittaci4 No + + + C pneumoniae - C pneumoniae 1:256 C pneumoniae5 No + ++ + C pneumoniae - C pneumoniae IgM + C pneumoniae6 No ± + ND - No significant titre C pneumoniae7 NK + + + + C pneumoniae - No significant titre C pneumoniae8 NK + + C pneumoniae - C pneumoniaelC psittaci

both > 1:256 C pneumoniae9 No + + C pneumoniae - Not available C pneumoniae10 NK + + C pneumoniae - C pneumoniae IgM + C pneumoniae11 Yes ± + ND - No significant titre C psittaci12 Yes ++ + Cpsittaci Cpsittaci Cpsittaci 1:16 -* 1:256 Cpsittaci13 Yes + + + + C pneumoniae - C psittaci 1:2048 C psittaci14 Yes + + C pneumoniae - No significant titre C psittaci15 NK - - / ND No significant titre16 NK - - / ND No significant titre -

17 NK - - / ND No significant titre18 NK - - / ND No significant titre19 NK + / ND No significant titre20 NK + / ND No significant titre21 Yes ± + IND C trachomatis No significant titre22 No ± + C trachomatis C trachomatis No significant titre23 Yes + + C psittaci Not available24 NK + + + ND No significant titre -

25 Yes + * + * ND * C pneumoniaelC psittaciboth IgM+ *

26 NK + + C psittaci Cpsittaci 1:4096

6 months after serum samples.

relevant clinical details and no other obviousaetiological cause was taken as suggestive ofrecent infection.

ResultsAll the 10 strains of C psittaci tested produceda first stage product of the expected size withno second stage amplification. The two Cpneumoniae strains were amplified in both thefirst stage and the second stage also withproducts of expected sizes (figure). None ofthe C trachomatis serovars and none of theother bacteria tested produced amplifications.Based on elementary body counts by DIF,the first round of PCR detected between 5 to10 particles and the second round detectedless than one elementary body of C pneumo-niae.Of the 26 sputum samples, PCR identified

10 as containing C pneumoniae, four as con-

taining C psittaci, and 12 as negative (table 2).Seven of 10 C pneumoniae strains identifiedby PCR were substantiated by serological evi-dence suggesting recent C pneumoniae infec-tion and all had evidence of chlamydialpresence by ELISA or DIF. Species specificDIF identified eight as C pneumoniae.Chlamydiae were isolated from case 3 but theorganism failed to grow on subsequent pas-sage and the isolate was consequently notidentified.PCR identified C psittaci in specimens from

four patients (cases 11-1 4). Two of these hadserological evidence of recent C psittaci infec-tion. The sputum from case 12 was also posi-tive by culture for C psittaci. Speciesdifferentiation by DIF suggested that strainsfrom cases 13 and 14 were C pneumoniaerather than C psittaci. However, both patientshad close bird exposure. In case 13 the sero-

logical profile taken over a four week period

after onset of illness showed a rise in C psittacititre up to 1/2048, whereas the correspondingtitre of C pneumoniae was only 1/64. Theblood sample of case 14 was taken too earlyto have clinically important species specificantibody titres.

Specimens from cases 15-18 had no evi-dence of chlamydial infection by ELISA or

DIF and no clinically important micro-IFresults. PCR identified all four as negative.Cases 19 and 20 were judged to be falselypositive by ELISA since there was no otherevidence of chlamydial infection. Cases 21and 22 were cases of C trachomatis. Theywere negative by PCR as expected since thestrategy used will not amplify DNA from Ctrachomatis. It was difficult to ascertainwhether the discrepant results in cases 23-25represented PCR false negatives or false posi-tive ELISA and DIF. Serum was unavailablefrom case 23, although he gave a history ofcontact with a sick parrot. Case 24 diedwithin the first week of illness, and the acuteserum did not have clinically importantmicro-IF titre. Case 25 had IgM antibodiesreacting with both C psittaci and C pneumo-niae. However, the sputum sample from thispatient was received six months after theblood sample when the patient presentedagain with recurrence of symptoms. It was

unclear whether he had had a persistentinfection or a separate episode of infectionwhich may or may not be chlamydial.Another blood sample from this patientreceived 18 months after the second episodeshowed a low titre of antibodies to C pneumo-niae with no detectable C psittaci titre. Case26 was probably a true C psittaci infection as

the micro-IF titre was very high, and the spu-tum sample was positive for antigen by bothELISA and DIF.

ND = Not done.NK = Not known.IND = Indeterminate.* Sputum sample received

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DiscussionIsolation is frequently taken as the gold stan-dard in microbiological practice. This maynot be valid with organisms that are difficultto grow. Of the 20 chlamydial culturesattempted in this series, four isolates weremade, of which only three (cases 12, 21, and22) were definitely identified. Two of thesethree isolates were C trachomatis. It is recog-nised that specimen handling proceduresused in the study were suboptimal for suc-cessful chlamydial cultivation. But the diffi-culty of isolating C pneumoniae from clinicalspecimens was well known.6 Development ofnucleic acid detection by PCR, which doesnot depend on organism viability, provides analternative method of diagnosis.C pneumoniae and C psittaci were the main

foci in this PCR study. C trachomatis, whichdoes not commonly cause pneumonia inadults and which is more easily diagnosed byconventional means, was excluded to makethe procedure and its interpretation simple.In the procedure described the sputum sam-ples required minimal treatment and nonucleic acid extraction steps were necessary.Mispriming was avoided by adopting the"Touchdown" PCR technique during thefirst stage.18 It was possible to completethe whole procedure within 24 hours bydoing one of the PCR stages overnight.

There have been reports of PCR inhibitorsbeing present in faeces, urine and eyeswabs.22 24 Inhibition was observed in twospecimens in this series (cases 11 and 12)during the initial development phase whenthe specimen preparation procedure consistedonly of a single heating step. The inclusion ofa proteinase K digestion step prior to heatingremoved the inhibitor, indicating its proteinnature in these two samples.

Titration studies and comparisons withDIF have suggested that this PCR approachwas very sensitive for C pneumoniae. Thespecificity appeared to be good with high cor-relation with micro-IF, ELISA, and DIF.Only one of the 10 patients (case 3) identifiedby PCR as C pneumoniae had a history of birdcontact, but results from both micro-IF andDIF suggested that the infection was with Cpneumoniae rather than C psiuaci. The sensi-tivity of the procedure for C psittaci wasinevitably lower due to the strategy chosen.The specificity was good, however, with highmicro-IF correlation. There were discrepan-cies with DIF in two specimens in which DIFidentified C pneumoniae; PCR detected Cpsittaci. Both patients had close bird exposureand micro-IF results supported PCR identifi-cation in one of the patients (case 13). Onbalance, the weight of evidence suggests thatDIF may have misidentified the chlamydialspecies in the sputum deposits of these twopatients. This problem of cross-reactivity ofthe C pneumoniae monoclonal antibody(Cellabs), which may partly be due to thespecimen treatment procedure, has beenreported before.25

Because the detection limit of the firststage PCR was believed to be between five

and 10 elementary bodies, cases containingonly a few C psittaci elementary bodies insputa could have been missed. The discrep-ancy between PCR and ELISA/DIF in cases23-26 could be explained by this reducedsensitivity. It is believed that PCR was falselynegative for the sputum sample from case 26.In case 24 only two elementary bodies weredetected by DIF in the first sputum smear,although a later specimen was more definitelypositive. In case 25 the clinical relevance ofthe serological findings was indeterminatebecause serum predated the sputum sampleby six months. Prolonged storage, repeatedfreeze-thaw cycles, and scanty specimensavailable for testing were other reasons whichmay have adversely affected PCR sensitivityin some of these samples. On the other hand,as both ELISA and the genus specific DIFused anti-lipopolysaccharide as the detectingantibodies, they could, theoretically, producesimilar false positive results.

Both the number of specimens and thedesign of the study prevented comparisonbetween the performance of ELISA, DIF,and PCR. There were two false positiveresults by ELISA in this series (cases 19 and20) and this suggested that direct visualisa-tion of elementary bodies is necessary to con-firm all ELISA positive results. However,DIF of sputum smears is difficult to readrequiring considerable experience particularlywhen there are only a few elementary bodiespresent. Diagnosis of C psittaci by the absenceof fluorescence staining with species specificantibodies is unreliable. PCR, however, offersa viable alternative.PCR, as developed in this study, seemed to

be both sensitive and specific for C pneumo-niae and also identified C psittaci with goodspecificity. Improvements in sensitivity for Cpsittaci can be made by introducing multiplexinternal primers which amplify both C pneu-moniae and C psittaci in the second stage. ThePCR method described can serve as a confir-matory test for screening based on ELISA orDIF. It can also be used as a tool to study therole and prevalence of C pneumoniae in spe-cial patient subgroups like asthmatics andthose with cystic fibrosis. Further studiescould define the role of PCR in routine clini-cal diagnosis.

This project was conducted with the help of a grant from theNewcasde Health Authority. Dr JD Treharne of the Instituteof Ophthalmology, London, kindly provided the variousstrains of Chlamydiae and performed micro-IF for the study.Professor C R Madeley, the supervisor of this project, and DrJSM Peiris, consultant virologist in Royal Victoria Infirmary,Newcastle Upon Tyne have given invaluable advice and guid-ance.

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