Major outbreak of pertussis in northern Alberta, Canada: analysis of discrepant direct fluorescent-antibody and culture results by using polymerase chain reaction methodology

Abstract

A major outbreak of 5,683 cases of pertussis occurred in northern Alberta, Canada, from December 1989 to January 1991. The outbreak highlighted a number of problems with current methods of pertussis diagnosis.In particular, an exceptionally high proportion of direct fluorescent-antibody (DFA)-positive, culture-negative specimens (88.4%) was identified. We took this opportunity to use polymerase chain reaction (PCR) methodology to examine whether the low culture rates were due to specimens containing dead organisms or whether the DFA results represented high numbers of false-positive results. A set of primer sequences within a Bordetella pertussis-specific repetitive element was used to amplify proteinase K extracts of B. pertussis DNA recovered from 279 submitted slides inoculated at the point of collection with nasopharyngeal material obtained from pernasal swabs. The PCR data corroborated the culture results: 84.6% of DFA-positive, culture-negative specimens were similarly PCR negative. At least three different bacterial species that were significantly cross-reactive with the commercial DFA reagent were identified in clinical specimens and in pure culture, providing one possible explanation for the false-positive DFA results. These results and other limitations of current diagnostic techniques underline the urgent need for a new DFA reagent with improved specificity and a standardized means of measuring the patient antibody response for the diagnosis of pertussis.Whooping cough (pertussis) may be 10 to 40 times more prevalent than passive reporting systems indicate (46). The true incidence of disease could be better determined if the means of diagnosing the disease was improved in North America (15,17,23,39). For one, the case definition of pertussis varies among different state and provincial health departments (8,15,27,42,46), which complicates reporting. For another, diagnosis itself is dramatically influenced by the time in the course of their disease that patients are seen. The clinical symptoms of pertussis are typically not unique early in the catarrhal stage (21,38,45) and the classic paroxysmal coughing that occurs later in the course of the disease may be absent, depending on the age of the patient, the patient's immune status, and whether the patient was treated with antibiotics (2,4,29,35,44).Laboratory diagnosis of pertussis is similarly affected by the same factors that influence symptomatology. Culture and fluorescent-antibody detection of organisms in nasopharyngeal swabs or aspirates are more likely to be positive just before or early after the onset of symptoms but are increasingly less likely to be positive as time elapses after the onset of cough (38,45,47). Conversely, serological tests, which are dependent on the new synthesis of pertussis-specific antibody in the patient, are more likely to be positive only after symptoms begin (29,32,45).In the province of Alberta, the case definition of pertussis is (i) characteristic paroxysmal cough, cough episodes ending in apnea, vomiting, or inspir...

Evaluation and validation of a monoclonal immunofluorescent reagent for direct detection of Bordetella pertussis

“…While in general this is true of our study as well, in our investigation the PCR assay was compromised by amplifying fixed material eluted from slides rather than sampling the clinical specimen directly. Indeed, a previous study demonstrated that the sensitivity of PCR drops 10-fold when it is performed with material eluted from slides rather than directly on suspensions of B. pertussis bacteria (9). In our study, there may have been an additional negative bias since the slides used for the PCR procedure were examined initially by the DFA test.…”

“…Following DFA analysis, a subset of 100 slides, including those with discordant findings relative to culture, were analyzed for the presence of B. pertussis DNA by PCR. The DNA was recovered from bacteria and cell debris smeared on the slides essentially as described previously (9), by resuspending the material in 50 l of 12 mM Tris-HCl (pH 7.6) and then transferring the suspension to a microcentrifuge tube. Proteinase K (Sigma Chemical Co., St. Louis, Mo.)…”

“…In the present study, we compared direct detection of B. pertussis in smears by fluorescein-conjugated MAb BL-5 with culture of nasopharyngeal swabs for 1,507 consecutive specimens sent to Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada, between 1 September and 1 November 1994. Additionally, PCR was used to resolve discordant findings for a subset of these specimens by amplifying B. pertussis DNA recovered from the slides after DFA analysis (9). In this report, we detail the validation of BL-5 in a clinical diagnostic setting and demonstrate the utility of this reagent for rapid and direct detection of B. pertussis.…”

“…The application of the PCR to pertussis diagnosis shows promise (9) but has not been sufficiently validated in a diagnostic setting (19). Direct detection of B. pertussis by fluorescein-conjugated-antibody (direct fluorescent-antibody [DFA]) staining can provide a rapid, presumptive diagnosis; however, several investigations have documented the lack of sensitivity and specificity of the existing commercially available, polyvalent immunological reagent (9,12,14,24). Extensive cross-reaction with normal nasopharyngeal and oral microbial floras makes the test technically difficult to interpret, resulting in false-positive rates as high as 85% (9).…”

“…Direct detection of B. pertussis by fluorescein-conjugated-antibody (direct fluorescent-antibody [DFA]) staining can provide a rapid, presumptive diagnosis; however, several investigations have documented the lack of sensitivity and specificity of the existing commercially available, polyvalent immunological reagent (9,12,14,24). Extensive cross-reaction with normal nasopharyngeal and oral microbial floras makes the test technically difficult to interpret, resulting in false-positive rates as high as 85% (9). Since these limitations might be addressed by using highly specific monoclonal antibodies (MAbs) for DFA analysis, we designed a study to evaluate a newly developed pertussis-specific MAb for direct detection of B. pertussis.…”