Laboratory diagnosis of Chlamydia pneumoniae infections

C HLA MY DIA PNEU MO NIAE IS A COM MON CAUSE OF RE SPIRA tory ill ness and ac counts for 10 to 20% of com mu nity ac quired pneu mo nia cases (1-3). Re cent se roepi de mi ol ogi cal stud ies have linked C pneu mo niae in fec tion with athe ro scle ro sis, asthma and acute ex ac er ba tions of chronic ob struc tive pul mo nary dis ease (4-6). In fec tion of ten ap pears as a mild, selflimiting ill ness but out breaks of C pneu mo niae in fec tion have been re ported within fami lies, in schools, mili tary bar racks and small com mu ni ties (7-12). In fec tion in preschoolaged chil dren are un com mon (13). Most in di vidu als ac quire their pri mary in fec tion of C pneu mo niae be tween five and 14 years of age. C pneu mo niae im mu no globu lin (Ig) G an ti bod ies may per sist for months to years, with less de cline in ti tre af ter re in fec tion than in pri mary in fec tion and in adults com pared with chil dren (13). Se ro preva lence stud ies con ducted world wide show that 50 to 70% of adults have IgG an ti bod ies RE VIEW

to C pneu mo niae, sug gest ing that re in fec tion is com mon through out adult hood.
The clini cal pres en ta tion of res pi ra tory ill ness caused by C pneu mo niae is of ten in dis tin guish able from that of vi ral or myco plasma eti ol ogy.Since cura tive an ti mi cro bial ther apy is avail able for C pneu mo niae in fec tion, spe cific labo ra tory di agno sis may be use ful for pa tient man age ment.In par ticu lar, where res pi ra tory ill ness is as so ci ated with a re cent his tory of bird or ani mal con tact, it is im por tant to rule out Chla my dia psit taci in fec tion, which may re quire pub lic health ac tion.In out break situa tions, a spe cific labo ra tory di ag no sis is im portant for early and ap pro pri ate in ter ven tion to pre vent fur ther spread of in fec tion.As C pneu mo niae be comes rec og nized as an im por tant emerg ing patho gen, rea gents for labo ra tory di ag no sis are now more widely avail able.An un der stand ing of the per form ance as well as limi ta tions of dif fer ent types of labo ra tory tests avail able for the di ag no sis of C pneu mo niae is es sen tial for the proper in ter pre ta tion of labo ra tory re sults.Ta ble 1 shows the at trib utes and limi ta tions of the di ag nos tic tests avail able for the labo ra tory di ag no sis of C pneu mo niae in fec tion.

CUL TURE
The iso la tion of C pneu mo niae in cell cul ture is tech ni cally more de mand ing than that of Chla my dia tra cho ma tis and often re quires mul ti ple pas sages over a pe riod of weeks in cell cul ture to show a posi tive re sult (14).Stud ies have shown that HEp-2 and HL cells are more sen si tive for C pneu mo niae than are HeLa or McCoy cells, which are tra di tion ally used for the cul ture of C tra cho ma tis (15)(16)(17).The use of serum-free me dia has been re ported to im prove iso la tion rates (18).The sen sitiv ity of cul ture is es ti mated to be 50% com pared with se rology.The speci fic ity is as sumed to be 100% be cause mono clonal an ti bod ies spe cific for C pneu mo niae are now com mer cially avail able for the iden ti fi ca tion of C pneu mo niae in clu sions in cell cul ture (19).Fail ure to iso late the patho gen may be due to sev eral rea sons such as in ade quate speci men sam pling, speci men tox ic ity in cell cul ture or fail ure to preserve speci men vi abil ity dur ing trans por ta tion of the specimen to the labo ra tory.Vi abil ity of the or gan ism is rap idly lost through freez ing and thaw ing.Speci mens from the throat and the na so pha rynx may have a lower yield of the or gan ism than deep-seated speci mens such as spu tum or bron choal veo lar lavage (BAL).Iso la tion from the na so pha rynx of healthy in dividu als has been re ported but the rate of as ymp to matic carriage in a nor mal popu la tion is un known (20,21).Hence, a posi tive iso la tion of C pneu mo niae from a non ster ile site should be in ter preted with cau tion.

AN TI GEN DE TEC TION TESTS
An ti gen de tec tion tests in clude di rect fluo res cent an ti body as says (DFA) and en zyme im mu no as says (EIA).These tests have the ad van tage of rapid turnaround time as well as ambi ent tem pera ture trans port.Speci mens taken for an ti gen de tec tion tests are sta ble at room tem pera ture for up to a week.For DFA, smears are usu ally made on site from na sopha ryn geal or throat swabs and fixed with ace tone or methanol be fore stain ing.The use of rayon swabs to col lect speci mens for DFA should be avoided be cause rayon fi bres left on the smear will auto fluo resce and in ter fere with read ing.Smears can be also be made from BAL speci mens but they may need to be di luted with sa line to mini mize non spe cific back ground stain ing.Metha nol was reported to de stroy the an ti genic re ac tiv ity of C pneu mo niae (22).Mon tal ban et al (19) evalu ated the use of sev eral C pneu mo niae-sp ecific fluo res cein-co nj ugated mono clonal an ti bod ies for stain ing in clu sions in cell cul ture and found that the choice of metha nol or ace tone as fixa tives de pended on the an ti body used.Results of smears can be avail able in 30 mins if an ti bod ies used for stain ing are di rectly con ju gated with fluo res cein.The sensi tiv ity of DFA is es ti mated to be 20 to 60% (23).A limi ta tion of DFA is that the read ing of the smear is sub jec tive.Hence, its speci fic ity is highly de pend ent on the ex per tise of the technolo gist.

TA BLE 1 Labo ra tory di ag no sis of Chla my dia pneu mo niae in fec tions
EIAs that are cur rently mar keted for C tra cho ma tis an ti gen de tec tion have been used for the de tec tion of C pneu mo niae from swabs (24,25).This is pos si ble be cause EIA tech nol ogy is based on the cap ture of the genus-specific chla my dial lipopoly sac cha ride (LPS).The sen si tiv ity of these EIAs is re ported to be com pa ra ble with that of DFA but, be ing LPS-based assays, they lack speci fic ity.Spu tum and BAL speci mens re quire mu co lytic di ges tion be fore be ing proc essed for EIAs (24).

NU CLEIC ACID-BASED TESTS
Po lymerase chain re ac tion (PCR) tech niques have been de vel oped for the de tec tion of C pneu mo niae DNA.A com parison of the pub lished pro ce dures for the labo ra tory de tec tion of C pneu mo niae is shown in Ta ble 2 (26)(27)(28)(29)(30)(31).Un like C tracho ma tis, C pneu mo niae does not pos sess a plas mid.Hence plasmid-based PCR kits for C tra cho ma tis can not be used to de tect C pneu mo niae.Spu tum, bron cho scopy speci mens, throat wash ings or swabs, and na so pha ryn geal swabs are suit able speci mens for PCR as says.Be cause DNA is sta ble in trans port, speci mens for PCR can be trans ported at room tempera ture if they can not be sent to the labo ra tory im me di ately.Traces of DNA may be pres ent up to three weeks af ter an ti biotic treat ment.
We evalu ated the user friend li ness and per form ance of four pub lished PCR pro to cols us ing 10-fold ti tra tions of C pneu mo niae strain TW-183 (32).The sen si tiv ity of all the proto cols was found to be com pa ra ble with de tec tion lim its of 10 to 100 ele men tary bod ies.The pro to cols of Camp bell et al (26) and Gay dos et al (28,30) were the most sen si tive and sim plest to per form be cause the prim ers are C pneu moniae-sp ecific and the speci fic ity of the am pli fied prod uct can be con firmed by probes in ter nal to the tar get se quence.The pro to col de vel oped by Tong and Sil lis (27) am pli fies a tar get se quence con served be tween C pneu mo niae and C psit taci and hence has the ad van tage of be ing able to de tect DNA from ei ther patho gen in a sin gle as say.A nested PCR pro ce dure is then used to dif fer en ti ate be tween the C pneu mo niae and C psit taci am pli cons.The pro to col of Ras mussen et al (29) ampli fies a genus-specific tar get se quence, fol lowed by spe cies dif fer en tia tion us ing re stric tion en zyme di ges tion.In col labora tion with the Pro vin cial Labo ra tory for South ern Al berta, we evalu ated 312 throat swabs col lected in 2-SP buffer as part of a vi ral watch pro gram (32).Three speci mens were culturepositive for C pneu mo niae in HeLa 229 cells.When the pro tocol of Camp bell et al was used, three swabs were PCR-positive, of which two were culture-positive.Fur ther evalua tions are on go ing.At pres ent, these PCR meth ods are re search tech niques.Mul ti plex PCR as says con tain ing prim ers spe cific for a panel of res pi ra tory patho gens are be ing de vel oped.

SE ROL OGY
The com ple ment fixa tion (CF) test has tra di tion ally been used for the se ro di ag no sis of res pi ra tory chla my dial in fections.The chla my dial an ti gen in volved in CF is the genusspecific LPS.There fore, the CF test can not be used to dis tinguish the an ti body re sponse re sult ing from C tra cho ma tis, C psit taci or C pneu mo niae in fec tions.The mi cro im mu no fluores cence (MIF) as say de vel oped by Wang et al ( 33) is used to de tect species-specific an ti bod ies and is the gold stan dard for chla my dia se rol ogy to day.An ti body cross-reactivity among chla my dia spe cies ob served in MIF may be due to the presence of an ti bod ies against genus-specific an ti gens such as the LPS or due to im pu ri ties in the an ti gen prepa ra tion (23,34,35).In sera from adults with an ti bod ies against C tracho ma tis and C pneu mo niae, it is likely that the pa tient has a his tory of in fec tion with both or gan isms.Be cause acute C pneu mo niae in fec tions gen er ally in duce higher lev els of IgG an ti body that are rarely seen in in fec tions with other chla mydia spe cies, low level cross-reactive an ti bod ies are un likely to pres ent a prob lem for the se ro di ag no sis of C pneu mo niae infec tions.Kits for EIA that use ex tracted or re com bi nant chlamy dial LPS and for MIF are com mer cially avail able.Al though less sen si tive than the MIF as say and lack ing in speci fic ity, both the CF test and the EIAs are much less tech ni cally demand ing than the MIF, are ame na ble to batch ing and have objec tive end-points.An other advan tage of these tests is that LPS an ti bod ies are pro duced early in in fec tion.Thus, a di ag no sis may be reached by the CF test or EIA in paired sera taken a week apart com pared with three weeks or more for MIF (36,37).There are also an ti body de tec tion kits where the an ti gens are cells in fected with a lym pho granu loma strain of C tra cho ma tis fixed onto a glass slide.Be cause an ti genic re lat ed ness between C pneu mo niae and lym pho granu loma has not been de ter mined, an ti body ti tres from these kits should be in terpreted with cau tion.

TA BLE 2 Com pari son of PCR pro ce dures for the de tec tion of Chla my dia pneu mo niae (Cpn) or Chla my dia psit taci (Cps)
The cri te ria for se ro posi tiv ity for past and acute in fec tion with C pneu mo niae are shown in Ta ble 3 (2).In col labo ra tion with Sas katche wan Health, we used these se ro posi tiv ity cri teria for MIF to evalu ate the use ful ness of the CF test in the di agno sis of C pneu mo niae in fec tions.Of 103 sera, the CF test was posi tive for four of seven (57%) sera posi tive for IgM an tibod ies by MIF against C pneu mo niae, but only de tected two of 16 (13%) sera posi tive for IgG an ti bod ies (38).Since IgM an tibod ies are rarely pro duced in re in fec tions with C pneu moniae, the CF test ap pears to be of lim ited use in the di ag no sis of re in fec tions in adults.Al though the num bers for our evaluation were small, these re sults were later con firmed in a much larger study.Ek man et al (36) com pared the per form ance of the CF, LPS-based EIA and MIF tests for the se ro di ag no sis of C pneu mo niae and C psit taci in fec tions in an eld erly popu la tion and found that the CF test has a sen si tiv ity of 10.3% com pared with 87.9% and 72.4% for MIF and EIA, re spec tively (36).IgM an ti bod ies were only de tected in 11.3% of cases.It is not clear why there was such dis par ity be tween the CF test and the EIA, both of which are LPS-based tests.
We re viewed the re sults of 2247 sera re ferred to the National Labo ra tory for Sexu ally Trans mit ted Dis eases for C pneu mo niae se rol ogy in 1994.Of 163 (7.8%) pa tients whose sera met the cri te ria for acute in fec tion, 27 (16%) were di agnosed based on IgM an ti body ti tre alone, four (2%) based on a four fold rise in IgG an ti body ti tre, and 132 (82%) on a com bina tion of IgM an ti body ti tre greater than 16 and IgG an ti body ti tre of 512 or greater (un pub lished data).The low per cent age of se ro di ag no sis by four fold rise in IgG an ti body ti tre may reflect the low number of paired sera (n=166) sent for test ing, insuf fi cient time be tween acute and con va les cent sera, or de lay in pa tients seek ing care, es pe cially if their symp toms were mild.The age of the those tested, the fre quency of re in fec tion and de lay in seek ing care may be re spon si ble for the low percent age of di ag no sis by IgM an ti bod ies.Se ro di ag no sis in the eld erly may be com pro mised by the pres ence of rheu ma toid fac tor and im mune se nes cence (39).The se ro logi cal response in chil dren ap pears vari able and needs fur ther evalua tion (23).

CON CLU SION
C pneu mo niae is an im por tant cause of res pi ra tory in fections.There is a need for more ac cu rate and rapid labo ra tory di ag nos tic meth ods that would im prove pa tient care through the ap pro pri ate use of an ti mi cro bial ther apy and ad vance our un der stand ing of the epi de mi ol ogy of these in fec tions.

Fea tures Camp bell et al (26) Tong & Sil lis (27) Gay dos et al (28,30) Ras mussen et al (29)
EB Ele men tary bod ies; PCR Po lymerase chain re ac tion PEELING 200 CAN J INFECT DIS VOL 6 NO 4 JULY/AUGUST 1995 CF Com ple ment fixa tion; EIA En zyme im mu no as say; Ig Im mu no globu lin; MIF Mi cro im mu no fluo res cence CAN J INFECT DIS VOL 6 NO 4 JULY/AUGUST 1995