Bordetella pertussis in the National Capital Region: prevalent ...
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Bordetella pertussis in the National Capital Region: prevalent serotype and immunization status of patients B. RossIER,* MD, DIP BACT (LOND); F. CHAN,t B SC, FIMLS Over a 2-year period 67 strains of Bordetella pertussis were identified in 231 single specimens of nasopharyngeal secretions submitted from patients suspected to have whooping cough in the National Capital Region; 89.50/0 of the identifications were made by culture. Serotype 1,3 was predominant. At least 750/0 of the patients with bacteriologically confirmed whooping cough had not been fully immunized. There was no evidence that adenoviruses or other viruses played any important etiologic role in the 204 cases of whooping cough or whooping cough syndrome studied virologically. Au cows d'une periode de 2 ans 67 souches de Bordetella pertussis ont ete identifiees a partir de 231 specimens de secretions nasopharyngees provenant de patients suspectes d'avoir Ia coqueluche dans Ia region de Ia Capitale nationale; 89.50/o des identifications ont ete faites en culture. Le serotype 1,3 etait predominant. Au moms 750/o des patients souffrant de coqueluche confirme bacteriolo. giquement navalent pas 6te completement vaccines. II n'a pas et. possible d'attribuer un role etiologique determinant aux adenovirus ou aux autres virus dans 204 cas de coqueluche ou de syndrome coqueluche etudies virologiquement. Virtually no isolations of Bordetella pertussis were made in the Ottawa re- gion before 1974, despite numerous notifications of cases of whooping cough to the Ottawa-Carleton Regional Health Unit: 81 in 1970, 114 in 1971, 35 in 1972, 46 in 1973 and 163 in 1974. This suggests that such cases might have been caused by adeno- viruses or other viruses. From Eleanor M. Paterson's department of laboratory medicine and research, Children's Hospital of Eastern Ontario, Ottawa *Professor, department of microbiology and immunology, faculty of medicine, University of Ottawa; head, regional virology laboratory, Eleanor M. Paterson's department of laboratory medicine and research, Children's Hospital of Eastern Ontario tLaboratory scientist, division of bacteriology, Eleanor M. Paterson's department of laboratory medicine and research, Children's Hospital of Eastern Ontario Presented in part at the 44th annual meeting of the laboratory division of the Canadian Public Health Association, Montreal, Dec. 1 to 3, 1976, in partial fulfilment of F. Chan's MSc thesis at the University of Ottawa Reprint requests to: Dr. E. Rossier, Department of microbiology and immunology, Faculty of medicine, University of Ottawa, Ottawa, Ont. KIN 9A9 Coincident with the opening of the Children's Hospital of Eastern Ontario in Ottawa in 1974, routine bacteriologic and virologic procedures were carried out with specimens collected from pa- tients suspected to have whooping cough. We report our laboratory find- ings for the period Jan. 1, 1975 to Dec. 31, 1976 Patients, specimens and methods Patients No attempt was made to select pa- tients in whom there was a high prob- ability that B. pertussis would be iso- lated. During the early part of this study, clinicians were unaware of our efforts to isolate B. pertussis, and ap- propriate cultures were set up simply because a diagnosis of whooping cough or whooping cough syndrome had been made on clinical grounds. Sixty-five percent of the specimens came from in- patients; in some instances specimens were obtained on several occasions, but only the results obtained from the ini- tial specimens have been considered in this study. Most patients (84%) were Ontario residents from the National Capital Region and did not live in institutions. There was no evidence of a cluster of cases associated with a particular school. Approximately the same number of patients lived in urban communities as in rural, and none were recent immigrants. Data on immunization status were gathered from the case histories and confirmed later through a questionnaire sent to the parents several weeks or months after the child's illness. It proved impossible to verify from the immunization records the type, origin or batch number of the vaccine given. However, since practically all vaccina- tions had been carried out by physi- cians in private practice in Ottawa, it can safely be assumed that the vaccine most often used was the Connaught combined diphtheria, pertussis, tetanus and poliomyelitis vaccine without ad- juvant. Complete immunization was de- fined as a primary course of three in- jections of B. pertussis vaccine and one or more booster injections. During the same period a large num- ber of inpatients and outpatients, most with a respiratory tract infection other than whooping cough or whooping cough syndrome, were investigated routinely at the regional virology labo- ratory. They served as a control group. Specimens Only nasopharyngeal secretions col- lected by Auger' suction, usually by nurses, were examined No transport medium was used and specimens were processed within 2 to 4 hours of col- lection. Methods Specimens were (a) stained by hema- toxylin-eosin and examined for the presence of ciliated cells, (b) examined by direct immunofluorescence for anti- gens of B. pertussis and B. parapertus- sis, (c) cultured for B. pertussis, B. parapertussis and other microorganisms and (d) inoculated into tissue cultures for virologic studies. Serotyping of the B. pertussis isolates was carried out in- dependently by three laboratories. Direct immunofluorescence: Difco reagents were used according to the method recommended by Difco Labo- ratories.' Slides were examined with two Leitz SM-LUX microscopes; a comparison bridge allowed simultaneous comparison between the test and the control slides. Ultraviolet source lights were HBO 50 W with KP500 filters. and incident illumination was used. Culture and identification: Specimens were inoculated on blood agar plates for common pathogens and on freshly prepared charcoal agar plates made from Oxoid medium CM 119 supple- mented with 10% horse blood, 1% Difco proteose peptone no. 3 and 35 p.g/mL of cephalexin. The charcoal agar plates were incubated for 7 days at 360C in a moist atmosphere contain- ing 5% CO2 and examined on days 3, 5 and 7 for the typical dew-like colonies of B. pertussis. Final identification was made by direct immunofluorescence as described above and by slide agglutina- tion with Difco Bacto B. pertussis and B. parapertussis antisera. Confirmatory biochemical tests were also carried out by two of the three outside laboratories prior to serotyping. Virologic studies: The suction tubes weue* washed out with culture medium and the specimens inoculated into tissue cLlltures of primary African green mon- key kidney and diploid human fetal lung. Hemadsorption tests were carried out on the 2nd day and repeated on the 10th day, and the cultures were examined for a cytopathic effect for CMA JOURNAL/NOVEMBER 19, 1977/VOL. 117 1169
Transcript of Bordetella pertussis in the National Capital Region: prevalent ...
Bordetella pertussis in the National Capital Region:prevalent serotype and immunization status of patientsB. RossIER,* MD, DIP BACT (LOND); F. CHAN,t B SC, FIMLS
Over a 2-year period 67 strains ofBordetella pertussis were identified in231 single specimens of nasopharyngealsecretions submitted from patientssuspected to have whooping coughin the National Capital Region; 89.50/0of the identifications were made byculture. Serotype 1,3 was predominant.At least 750/0 of the patients withbacteriologically confirmed whoopingcough had not been fully immunized.There was no evidence thatadenoviruses or other viruses playedany important etiologic role in the204 cases of whooping cough orwhooping cough syndrome studiedvirologically.
Au cows d'une periode de 2 ans67 souches de Bordetella pertussis ontete identifiees a partir de 231 specimensde secretions nasopharyngeesprovenant de patients suspectes d'avoirIa coqueluche dans Ia region de IaCapitale nationale; 89.50/o desidentifications ont ete faites en culture.Le serotype 1,3 etait predominant.Au moms 750/o des patients souffrantde coqueluche confirme bacteriolo.giquement navalent pas 6tecompletement vaccines. II n'a pas et.possible d'attribuer un role etiologiquedeterminant aux adenovirus ou auxautres virus dans 204 cas decoqueluche ou de syndrome coquelucheetudies virologiquement.
Virtually no isolations of Bordetellapertussis were made in the Ottawa re-gion before 1974, despite numerousnotifications of cases of whoopingcough to the Ottawa-Carleton RegionalHealth Unit: 81 in 1970, 114 in 1971,35 in 1972, 46 in 1973 and 163 in1974. This suggests that such casesmight have been caused by adeno-viruses or other viruses.
From Eleanor M. Paterson's department oflaboratory medicine and research, Children'sHospital of Eastern Ontario, Ottawa*Professor, department of microbiology andimmunology, faculty of medicine, Universityof Ottawa; head, regional virology laboratory,Eleanor M. Paterson's department of laboratorymedicine and research, Children's Hospital ofEastern OntariotLaboratory scientist, division of bacteriology,Eleanor M. Paterson's department of laboratorymedicine and research, Children's Hospital ofEastern Ontario
Presented in part at the 44th annual meetingof the laboratory division of the CanadianPublic Health Association, Montreal, Dec. 1 to 3,1976, in partial fulfilment of F. Chan's MScthesis at the University of Ottawa
Reprint requests to: Dr. E. Rossier, Departmentof microbiology and immunology, Faculty ofmedicine, University of Ottawa, Ottawa, Ont.KIN 9A9
Coincident with the opening of theChildren's Hospital of Eastern Ontarioin Ottawa in 1974, routine bacteriologicand virologic procedures were carriedout with specimens collected from pa-tients suspected to have whoopingcough. We report our laboratory find-ings for the period Jan. 1, 1975 to Dec.31, 1976
Patients, specimens and methods
PatientsNo attempt was made to select pa-
tients in whom there was a high prob-ability that B. pertussis would be iso-lated. During the early part of thisstudy, clinicians were unaware of ourefforts to isolate B. pertussis, and ap-propriate cultures were set up simplybecause a diagnosis of whooping coughor whooping cough syndrome had beenmade on clinical grounds. Sixty-fivepercent of the specimens came from in-patients; in some instances specimenswere obtained on several occasions, butonly the results obtained from the ini-tial specimens have been considered inthis study. Most patients (84%) wereOntario residents from the NationalCapital Region and did not live ininstitutions. There was no evidence ofa cluster of cases associated with aparticular school. Approximately thesame number of patients lived in urbancommunities as in rural, and none wererecent immigrants.
Data on immunization status weregathered from the case histories andconfirmed later through a questionnairesent to the parents several weeks ormonths after the child's illness. Itproved impossible to verify from theimmunization records the type, originor batch number of the vaccine given.However, since practically all vaccina-tions had been carried out by physi-cians in private practice in Ottawa, itcan safely be assumed that the vaccinemost often used was the Connaughtcombined diphtheria, pertussis, tetanusand poliomyelitis vaccine without ad-juvant. Complete immunization was de-fined as a primary course of three in-jections of B. pertussis vaccine and oneor more booster injections.
During the same period a large num-ber of inpatients and outpatients, mostwith a respiratory tract infection otherthan whooping cough or whoopingcough syndrome, were investigated
routinely at the regional virology labo-
ratory. They served as a control group.
Specimens
Only nasopharyngeal secretions col-lected by Auger' suction, usually bynurses, were examined No transportmedium was used and specimens wereprocessed within 2 to 4 hours of col-lection.
Methods
Specimens were (a) stained by hema-toxylin-eosin and examined for thepresence of ciliated cells, (b) examinedby direct immunofluorescence for anti-gens of B. pertussis and B. parapertus-sis, (c) cultured for B. pertussis, B.parapertussis and other microorganismsand (d) inoculated into tissue culturesfor virologic studies. Serotyping of theB. pertussis isolates was carried out in-dependently by three laboratories.
Direct immunofluorescence: Difcoreagents were used according to themethod recommended by Difco Labo-ratories.' Slides were examined withtwo Leitz SM-LUX microscopes; acomparison bridge allowed simultaneouscomparison between the test and thecontrol slides. Ultraviolet source lightswere HBO 50 W with KP500 filters.and incident illumination was used.
Culture and identification: Specimenswere inoculated on blood agar platesfor common pathogens and on freshlyprepared charcoal agar plates madefrom Oxoid medium CM 119 supple-mented with 10% horse blood, 1%Difco proteose peptone no. 3 and 35p.g/mL of cephalexin. The charcoalagar plates were incubated for 7 daysat 360C in a moist atmosphere contain-ing 5% CO2 and examined on days 3,5 and 7 for the typical dew-like coloniesof B. pertussis. Final identification wasmade by direct immunofluorescence asdescribed above and by slide agglutina-tion with Difco Bacto B. pertussis andB. parapertussis antisera. Confirmatorybiochemical tests were also carried outby two of the three outside laboratoriesprior to serotyping.
Virologic studies: The suction tubesweue* washed out with culture mediumand the specimens inoculated into tissuecLlltures of primary African green mon-key kidney and diploid human fetallung. Hemadsorption tests were carriedout on the 2nd day and repeated onthe 10th day, and the cultures wereexamined for a cytopathic effect for
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14 days. Cultures with positive resultswere further identified by complementfixation and neutralization tests and byelectron microscopy. Viral serology wasnot performed because an early bloodspecimen was not available in mostcases.
Results
Quality of the specimens
Only 21% of the specimens con-tained ciliated cells, which suggestspoor sampling technique consideringthat 65% of the specimens were col-lected in the catarrhal stage of the dis-ease. At this stage there is desquama-tion and necrotizing inflammation ofthe superficial epithelial layers of theentire respiratory tract.3
Identification of B. pertussis in relationto age and immunization status of thepatients (Table I)Of the 231 Auger suction specimens
from patients with a clinical diagnosisof whooping cough or whooping coughsyndrome, B. pertussis was identified in67 (29%); 89.5% of the identificationswere made by culture and the othersby direct immunofluorescence. While12 identifications (17.9% of the 67)were made in completely immunizedchildren, at least half were made inpatients who were either less than 1year old (53%) or incompletely im-munized (61 %). Among those whowere incompletely immunized 15 (22%)were less than 3 months old and there-fore could not have been expected tohave received any vaccine.The three isolations of B. paraper-
tussis made during the period of studyare not included in the tables.
Identification of B. pertussis in relationto onset of symptoms and antibiotictreatment (Table II)
In the six instances in which onlythe immunofluorescence test resultswere positive, antibiotics (penicillin, am-picillin or erythromycin) had been ad-ministered. Few isolations were madebeyond the 5th week of the disease,irrespective of the administration of anantibiotic. However, 16 identificationswere made during the paroxysmal stageof the disease.Of the patients with negative results
of culture for B. pertussis 57.1% hadreceived antibiotics, as compared with24.6% of those with positive results -a significant difference (P < 0.001)that may account for the negative cul-tures in 151 instances.
Comparison of results of directimmunofluorescence and culture(Table III)Enough material was available in 192
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Auger suction specimens to allowproper comparison between the resultsfor the two types of test. Of 52 speci-mens yielding positive results on cul-ture, nearly half (24) gave negativeresults on direct immunofluorescence,whereas of 140 specimens yieldingnegative results on culture only 6 gavepositive results on direct immunofluor-escence. Hence the identification rateis increased by 11 % if cultures areperformed in conjunction with directimmunofluorescence. Conversely, therate would have decreased by 13%if direct immunofluorescence alone hadbeen used.
Virologic studies (Table IV)Virologic investigations were carried
out simultaneously on nasopharyngealsecretions from 204 patients with adiagnosis of whooping cough or whoop-ing cough syndrome and from 1621other patients, most with a respiratorytract infection other than whoopingcough.Th& proportion of virus isolations in
the cases of whooping cough in whichB. pertussis was not isolated was notsignificantly different from the propor-tion in the cases in which B. pertussiswas isolated. The proportion of virusisolations in the cases of whoopingcough was only marginally significantlydifferent from the proportion in thecases of other respiratory tract infec-tions (X2 = 2.27, P = 0.20).
Serotypes of B. pertussisThe three independent laboratories
found all 55 isolates of B. pertussis intheir second to fourth subculture to beof serotype 1,3.
Discussion
Our study has demonstrated thepresence of B. pertussis in the NationalCapital Region. The rate of isolationof the organism increased from 0in 439 patients with whooping coughbefore 1974 to 67 in 231 since 1974.In conditions similar to ours B. per-tussis can be isolated from a singlespecimen of nasopharyngeal secretionsin about one of every three suspectedcases of the disease. Although mostisolations were made in the catarrhalstage, a substantial proportion weremade beyond the 2nd week of the ill-ness, even when antibiotics had beengiven. Contrary to several reports,4-6 wefound that direct immunofluorescencewas far less sensitive a technique thanculture with the use of commerciallyavailable reagents. The high proportionof false-positive results with the formeris also a problem.7 Considering theusual poor quality of our material andthe fact that only single specimens were
studied we have little doubt that B.pertussis could be identified in nearly50% of the cases if better specimenswere obtained.
In a similar study Islur, Anglin andMiddleton8 reviewed the findings in 251children admitted to the Hospital forSick Children in Toronto for suspectedwhooping cough. B. pertussis was cul-tured from nasopharyngeal secretionscollected at the time of admission andon days 2, 4, 6 and 10 from 175 ofthe children. However, as impressiveas that figure might be, clearly quintu-plicate sampling is prohibitively ex-pensive and impossible to do in dailypractice. We attribute the good resultsobtained with single specimens to thefact that they were cultivated on char-coal agar plates containing 35 ,.Lg/mLof cephalexin rather than on Bordet-Gengou plates containing 0.25 U/mLof penicillin G. We have shown thaton the latter medium a few colonies ofB. pertussis can easily be overgrownby throat commensals or inhibited bythis concentration of penicillin G (F.Chan and E. Rossier: unpublished data,1977).
Viruses have been suggested as acause of whooping cough syndrome.9'10There was only a marginally significantdifference in the virus isolation ratebetween the whooping cough cases andthe cases of other respiratory tract in-fections, and we found no clear-cutevidence for a viral cause in the 151cases of whooping cough in which theresults of culture for B. pertussis werenegative. Antibiotic treatment was morelikely responsible for our negative cul-tural findings. Similarly, in the Torontostudy,8 though adenoviruses were iso-lated in 10% and respiratory syncytialvirus in 4% of cases in which resultsof culture for B. pertussis were positive,these viruses were not considered to bean important etiologic factor in thewhooping cough syndrome. In bothstudies at least 75% of the patientswith proven whooping cough had notbeen fully immunized against thedisease.
In many countries over the pastfew years B. pertussis serotypes havechanged from a mixture of types 1, 2,3 and 1,2 to a predominance of type1,3. This change is related to the intro-duction of mass vaccination with vac-cines rich in antigens 1 and 2 but weakin or devoid of antigen 3. Furthermorethere have been outbreaks of type 1,3infections in fully vaccinated children.11The National Capital Region is no ex-ception: type 1,3 is also the prevalentserotype here, in keeping with the find-ings of Anglin and Fleming (personalcommunication, 1977) and others inOntario12'13 and Nova Scotia.1'
Conclusions
Immunization against whoopingcough was introduced in Canada in the1940s and thereafter the reported in-cidence of the disease decreased.14 Theextent to which immunization, as com-pared with other less specific factors,has played a decisive role in thisdecrease is debatable. As in othercountries'5 there is controversy in Can-ada about the need for and the safetyand efficacy of pertussis vaccination.Apart from the well known underre-porting of the disease there are remark-ably few laboratory data availableabout the true incidence of B. pertussisand its serotypes across the country, al-though our investigation has demon-strated that they can be obtained infield conditions. Such data are neces-sary before a rational policy for oragainst pertussis vaccination can beestablished in Canada.
We thank Dr. N.W. Preston, Universityof Manchester, England, Dr. S. Toma andMr. M. Magus, central laboratories, On-tario Ministry of Health, Toronto and Dr.J. Cameron, Connaught Laboratories, Ltd.,Toronto for kindly serotyping our isolates.We also thank Mr. P.H. Phipps, laboratoryscientist, regional virology laboratory,Children's Hospital of Eastern Ontario,Ottawa and the laboratory's staff for valu-able assistance.
References
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11. PRESTON NW: Prevalent serotypes of Borde-tella pertussis in non-vaccinated communities.i Hyg 77: 85, 1976
12. CHALvARDJIAN N: The content of antigens1, 2 and 3 in strains of Bordetella pertussisand in vaccines. Can Med Assoc J 92: 1114,1965
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