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0
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1996, American Society for Microbiology
Multiplex PCR-Based Assay for Detection of Bordetella pertussis
in Nasopharyngeal Swab Specimens
ROBERT M. WADOWSKY,
1,2,3* RICHARD H. MICHAELS,
3,4THERESE LIBERT,
3LAWRENCE A. KINGSLEY,
2ANDGARTH D. EHRLICH
1,2,3,5Departments of Otolaryngology,
5Pathology,
1and Pediatrics,
4School of Medicine, and Department of Infectious
Diseases and Microbiology, Graduate School of Public Health,
2University of Pittsburgh, Pittsburgh, Pennsylvania
15261, and Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213
3Received 19 April 1996/Returned for modification 16 July 1996/Accepted 8 August 1996
A multiplex PCR-based assay was developed for the detection of
Bordetella pertussis
in nasopharyngeal swab
specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a
B. pertussis
repetitive element and a 438-bp target within the
b
-actin gene of human DNA (PCR amplification control).
PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496
paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30
(6%) of the specimens inhibited the amplification of the
b
-actin target, in all 29 specimens studied, the
inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest.
Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the
PCR-based assay, whereas 13.9% were positive by culture (
P
< 0.0001). After resolving the PCR-positive,
culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based
assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with
values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed
pertus-sis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough,
immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is
substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.
The recent increase of pertussis in the United States (7)
underscores the need for rapid and accurate diagnostic
meth-ods to guide therapeutic and preventive interventions and to
assess the effectiveness of acellular vaccines. Laboratory
diag-nosis of pertussis is usually accomplished by culture, which
requires special media and incubation for up to 7 days and
which is insensitive, especially when nasopharyngeal secretions
are obtained outside the early catarrhal stage of the illness
(25), from vaccinated persons (3, 12), or from persons treated
with certain antibiotics (3, 5). Direct fluorescent-antibody
test-ing provides rapid results but is even less sensitive than culture
and not as specific (8). Although serological testing is useful
for epidemiological studies, it is not used for routine diagnosis
since a high degree of sensitivity depends on measuring
anti-body responses to multiple antigens, acute- and
convalescent-phase sera are usually required, and antibody titers resulting
from maternal transfer or response to vaccine complicate
in-terpretation (12, 16, 26).
Previous studies suggest that PCR-based assays for the
de-tection of Bordetella pertussis in nasopharyngeal specimens are
sensitive and specific (2, 9, 23, 27) and are superior to culture
for identifying mild illness caused by B. pertussis (23).
PCR-based assays that amplify targets within the B. pertussis
repet-itive element, which is present in 50 to 100 copies in B. pertussis
chromosomal DNA (9), may be more sensitive than those that
amplify single-copy targets (19). Further improvements in the
PCR-based assays could include the use of an internal positive
control system to identify specimens that inhibit the PCR (28,
30); the amplification of multiple rather than single B. pertussis
DNA targets to provide an increased assurance of specificity,
since carryover contamination from each amplimer is thought
to occur independently (24); the use of uracil-N
9
-glycosylase to
degrade contaminating amplimeric DNA (22); and the use of
a sensitive and specific postamplification hybridization test for
confirmation of the PCR products (24).
In the present study we evaluated a multiplex PCR-based
assay for the detection of B. pertussis in nasopharyngeal swab
specimens. The assay simultaneously amplifies two separate
DNA targets within the repetitive element of B. pertussis DNA
and a third target within the
b
-actin gene of human DNA
(PCR amplification control). The PCR products are detected
by a liquid hybridization gel retardation assay (24).
MATERIALS AND METHODS
Strains and cell culture.Recent clinical isolates of bacteria and yeasts were obtained from the clinical microbiology laboratory of Children’s Hospital of Pittsburgh (Pittsburgh, Pa.). Reference strains were obtained from the American Type Culture Collection (ATCC; Rockville, Md.). B. pertussis BP5 was isolated from the nasopharynx of a child with pertussis and was grown on charcoal agar (Oxoid Unipath Ltd., Basingstoke, England) containing 10% defibrinated sheep blood. Strains of other microorganisms were selected to evaluate the specificity of the B. pertussis primer-probe sets including Bordetella parapertussis ATCC 9305, ATCC 15311, ATCC 15237, ATCC 15989, and BP181 (clinical isolate); Bordetella bronchiseptica ATCC 19, ATCC 780, ATCC 785, ATCC 786, and ATCC 10580; Bordetella avium ATCC 35086; and the following strains isolated from throat swab specimens obtained from children with upper respiratory tract infection: 6 Candida albicans, 6 Corynebacterium spp., 7 Haemophilus influenzae, 5 Haemophilus parainfluenzae, 6 Moraxella catarrhalis, 6 Neisseria spp. other than Neisseria meningitidis and Neisseria gonorrheae, 6 Staphylococcus aureus, 6 coag-ulase-negative Staphylococcus spp., 10 Streptococcus pyogenes, 6 Streptococcus pneumoniae, 7 viridans group Streptococcus spp., and 1b-hemolytic Streptococcus sp. (not group A). A lymphoblastoid cell culture line, CEM, was obtained from the Molecular Diagnostics Laboratory (University of Pittsburgh, Pittsburgh, Pa.).
DNA isolation.Cells were digested with lysozyme and proteinase K, and the chromosomal DNA was purified by extraction with phenol-chloroform-isoamyl alcohol and precipitation with ethanol as described previously (29).
Patients and samples.Specimens from children who were evaluated for
per-* Corresponding author. Mailing address: Department of Pathology,
Children’s Hospital of Pittsburgh, 3705 Fifth Avenue at Desoto Street,
Pittsburgh, PA 15213. Phone: (412) 692-5314. Fax: (412) 692-6550.
2645
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tussis at the Children’s Hospital of Pittsburgh between July 1992 and June 1995 were included in the study, which was approved by the hospital’s institutional review board. Nasopharyngeal swab specimens for culture and the PCR-based assay were obtained with calcium alginate and Dacron swabs (Medical Packaging Corp., Camarillo, Calif.), respectively, since calcium alginate swabs are superior to Dacron swabs for the isolation of B. pertussis (8, 13) and calcium alginate swabs contain substances that inhibit a PCR-based assay, whereas Dacron swabs do not (28). The order of collection with the swab types was alternated between patients, and the specimens were obtained as described previously (8). The specimens on calcium alginate swabs were inoculated at the side of the patient onto freshly prepared (i.e., on the day of usage) Bordet-Gengou agar plates containing 20% sheep blood and 0, 5, and 40mg of cephalexin per ml. Specimens on Dacron swabs were placed into 12-by-75-mm polypropylene vials containing 0.5 ml of saline (Medical Packaging Corp.). These materials were immediately transported to the laboratory for processing. The contents of the transport vial were vortexed for 10 s, and the swab was then wrung against the inside wall of the vial to express excess moisture and was discarded. Aliquots (0.1 ml) of the suspension were stored at2808C for subsequent testing by the PCR-based assay. The inoculum on the agar plates was streaked for isolation, and the plates were incubated at 358C for 7 days in a moist chamber (1). The identification of B. pertussis was confirmed by agglutination with B. pertussis-specific antisera and no agglutination with B. parapertussis-specific antisera (Difco Laboratories, Detroit, Mich.). B. parapertussis was not isolated from any of the patients. Specimens were not evaluated for the presence of B. bronchiseptica.
Multiplex PCR-based assay.Control swabs were prepared by using pooled samples of nasopharyngeal aspirates obtained from children with suspected respiratory syncytial virus infection who had tested negative for B. pertussis by the multiplex assay. A washed suspension of B. pertussis BP5 (28) was added to a portion of the pooled samples to provide about 104CFU/ml. Dacron swabs were dipped into the seeded and unseeded samples to prepare positive and negative control swabs, respectively (28). The control swabs were placed into the transport tubes and were processed in the same way as the clinical specimens.
(i) Oligonucleotides.Oligonucleotides were synthesized with an automated DNA synthesizer (model 391; Applied Biosystems, Inc., Foster City, Calif.) by using standardb-cyanoethyl phosphoramidite chemistry. Primers HAC3 and HAC5 define a 438-bp target within theb-actin gene of human DNA (11, 20). Probe HAC-PR was designed to detect the PCR product. The 59339nucleotide sequence of HAC-PR (positions 2074 to 2106) was GAGCGCGGCTACAGCT TCACCACCACGGCCGAG. Primers BP1 and BP2 (our designation for the primers published by Glare et al. [9] and Wadowsky et al. [29]) and primers BP13 and BP14 define 153 and 203-bp targets, respectively, within the B. pertussis repetitive element (9, 28, 29). DNA probes BP3 and BP15 are complementary to internal sequences within the respective DNA targets (28, 29). The probes were end labeled with32P by using T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.) (18).
(ii) PCR amplification.DNA was amplified in 100-ml reaction mixtures con-taining 200mM (each) dATP, dUTP, dCTP, and dGTP; 2.0 U of Amplitaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.); 1.0 U of uracil-N9 -glycosy-lase (Life Technologies Inc., Gaithersburg, Md.); 0.1mM (each) primers BP1, BP2, BP13, and BP14; 0.25mM (each) primers HAC3 and HAC5; 3.75 mM MgCl2; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 10 ng of CEM DNA (included as an optimal amount of template for the HAC3 and HAC5 primers since swab specimens do not provide a reliable target); and 50ml of sample. Frozen samples were thawed and digested with proteinase K as described previously (9, 28). Sample digests were tested in duplicate. Each run included a positive and a negative control digest, a reagent control for every five specimens, saline from a transport tube, a B. pertussis DNA control dilution series (1 pg and 100, 50, 10, and 5 fg), and Tris-EDTA buffer (29), which was used to dilute the DNA. Thermal cycling conditions were identical to those published previously (28). The criteria for the acceptability of a run included the following: obtaining the expected results with the positive, negative, and reagent controls and obtaining a lower limit of detection of at least 50 fg of B. pertussis genomic DNA. Results from runs not meeting these criteria were rejected and the test was repeated.
(iii) Detection and analysis of PCR products.The PCR products were simul-taneously hybridized in solution with the32
P-labelled probes, and the hybrid molecules were separated by electrophoresis through a 5% polyacrylamide gel and detected by autoradiography (29). Testing of specimen digests in duplicate provided a maximum of two bands corresponding to the 438-bpb-actin gene product and four bands corresponding to the 153- and 203-bp B. pertussis prod-ucts. Specimens yielding two bands corresponding to theb-actin gene product were considered evaluable and were then interpreted with respect to the bands corresponding to the B. pertussis DNA products as follows: positive, three or four bands; indeterminate, two bands; and negative, no bands or one band. Specimens with indeterminate results were retested, and those results were considered final. Specimens that yielded either no band or one band corresponding to theb-actin gene product were initially interpreted as nonevaluable and were further evalu-ated for inhibitory substances by digesting specimen aliquots with proteinase K, diluting the digests in Tris-EDTA buffer in twofold steps up to 1:16, and testing the samples by the PCR-based assay. This additional testing always yielded evaluable results, which were interpreted as described above.
All specimen preparations, PCR setups, and PCR amplifications were per-formed in separate laboratories that were designed and staffed to minimize end product contamination (24).
Analysis of discrepant results.The sensitivities and specificities of the PCR-based assay and culture were determined as described by Loeffelholz et al. (15) following the resolution of discrepant results. PCR-negative, culture-positive results from specimen pairs indicated a false-negative result for the PCR-based assay and a true-positive result for culture. PCR-positive, culture-negative results were resolved by repeating the PCR-based assay with a frozen aliquot of the specimen suspension. If the repeat test was positive, the initial PCR result was considered a true-positive result and the culture result was considered a false-negative result. If the repeat test was false-negative, the initial PCR result was con-sidered a false-positive result and the culture result was concon-sidered a true-negative result. Our approach for resolving the PCR-positive, culture-true-negative results excludes the possibility that the initial PCR result occurred from car-ryover contamination with amplimeric DNA during the preparation of the PCR mixture.
Review of medical records.The hospital charts were reviewed for the age of the patient, immunization status, whether antibiotics were taken prior to testing, duration of cough, presence of paroxysms, posttussive emesis or inspiratory whoop, and results of viral culture, if one was performed. In some cases, patients were sent to the hospital for laboratory tests only, and no clinical information was available in the hospital chart. Relevant data, however, were usually available from the office records of the referring physicians.
Statistical analysis.The proportions of patients with culture-positive and PCR-positive results for B. pertussis were treated as paired, dichotomous vari-ables and were analyzed by McNemar’s test. The sensitivity analysis comparison of PCR and culture positivity was performed by a test of two proportions, in reference to the z statistic. Differences in the isolation rates of viruses and in the proportions of patients who received erythromycin or three or more doses of pertussis vaccine, were$2 years of age, or had coughed for$1 month were analyzed by thex2
test and Fisher’s exact test. P values were determined with the Epistat software program (10).
RESULTS
Analytical sensitivity and specificity.
Purified genomic
DNAs were used to evaluate the sensitivity and specificity of
the multiplex PCR-based assay. The effect of excess human
DNA on the amplification of the two B. pertussis DNA targets
was evaluated by testing the B. pertussis DNA control series in
PCRs with and without 1.0
m
g of CEM DNA (Fig. 1). Both B.
pertussis PCR products (i.e., 153 and 203 bp) were detected in
reaction mixtures containing 10 fg (about two genomic copies)
of B. pertussis DNA in the presence and absence of the CEM
DNA (Fig. 1). Therefore, the assay achieved nearly single-copy
detection of B. pertussis DNA even in the presence of excess
human DNA.
To assess the specificities of the two B. pertussis
primer-probe sets, 10 ng (
.
10
6genomic equivalents) of DNAs from 83
isolates of bacteria and yeasts were tested in the assay with only
these primer-probe sets and no input CEM DNA. None of the
DNAs yielded detectable PCR products, demonstrating the
high degree of specificity for the primer-probe sets.
Detection of
B. pertussis
in nasopharyngeal swab specimens.
The testing of selected nasopharyngeal swab specimens
indi-cated that the assay could discriminate specimens that were
FIG. 1. Autoradiograph showing the effect of excess human DNA on the lower limit of detection of B. pertussis by the multiplex PCR-based assay. One copy of B. pertussis DNA corresponds to about 5 fg of purified genomic DNA.on May 15, 2020 by guest
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either culture positive or culture negative for B. pertussis and
identify specimens that may contain inhibitors of PCR (Fig. 2).
A total of 496 paired swab specimens were obtained from 486
patients and were independently tested for B. pertussis by the
multiplex PCR-based assay and culture.
(i) Specimens with possible inhibitory substances.
A
non-evaluable result was initially obtained by the PCR-based assay
for 30 (6%) of the specimen pairs. Only one of these pairs
yielded growth of B. pertussis, and the density was heavy.
Fro-zen aliquots, available from 29 of these pairs, were assessed for
PCR inhibitors by testing undiluted and diluted sample lysates.
Testing of the undiluted lysates yielded a positive result for 2
specimens, an indeterminate result for 1 specimen, a negative
result for 16 specimens, and a nonevaluable result for 10
spec-imens. One of the two specimens yielding the positive results
by the PCR-based assay was from the patient with the positive
culture result. Of the 10 specimens yielding the nonevaluable
results from the undiluted samples, 6 were negative at a 1:2
dilution, 3 were negative at a 1:4 dilution, and 1 was negative
at a 1:8 dilution. These results suggest that specimens yielding
nonevaluable results contain low levels of inhibitory substances
and that either repeat testing of the undiluted specimen or
testing following a small dilution can provide a final evaluable
result.
(ii) PCR versus culture.
We next compared the results of the
PCR-based assay and culture for the 495 specimen pairs (i.e.,
99.8% of the total pairs), including those specimens assessed
for inhibitors, yielding a final evaluable result by the
PCR-based assay. Positive results were obtained by both assays for
68 pairs, and negative results were obtained by both assays for
394 pairs. One pair yielded PCR-negative, cultupositive
re-sults. This culture yielded a light growth of B. pertussis.
PCR-positive, culture-negative results were obtained for 26 pairs.
The PCR-based assay yielded an indeterminate result for six
pairs (all were culture negative). The proportion of pairs with
PCR-positive results (i.e., 19.0%) was significantly greater than
the proportion of pairs with culture-positive results (i.e.,
13.9%) (P
,
0.0001).
Since indeterminate results by the PCR-based assay were
considered to be neither positive nor negative, they were
ex-cluded from the analysis of sensitivity and specificity, providing
489 evaluable pairs (i.e., 99% of the total number). Repeat
testing of specimen aliquots for the 26 pairs with PCR-positive,
culture-negative results by the multiplex PCR-based assay
yielded positive results for 25 patients. The sensitivities of the
PCR-based assay and culture were therefore 98.9 and 73.4%,
respectively (P
,
0.001) (Table 1). The specificity of the
PCR-based assay was 99.7%, compared with a specificity of 100%
(by definition) for culture.
Comparison of patients with culture-confirmed pertussis
and PCR-positive, culture-negative results.
Medical records
were reviewed to identify differences between the 25 patients
with the PCR-positive (confirmed), culture-negative results
and 82 patients with culture-confirmed pertussis. This group of
patients with culture-confirmed pertussis contains additional
patients from whom a specimen for PCR testing was not
ob-tained. A strong temporal relationship was observed between
the two groups. For example, the majority of cases occurred in
1993 in the Pittsburgh area (i.e., 55 [67%] patients in the
culture-confirmed group and 17 [71%] patients in the
PCR-positive, culture-negative group).
The following viruses were isolated from the nasopharynges
of 66 study patients: respiratory syncytial virus, n
5
46;
influ-enza type A or B virus, n
5
9; parainfluenza virus, n
5
5;
enterovirus, n
5
5; and cytomegalovirus, n
5
1. Virus was
detected in 3 of 82 (4%) patients with culture-confirmed
per-tussis and in a similar proportion of PCR-positive,
culture-negative patients (i.e., 1 of 24). In contrast, virus was isolated
significantly more often from the group of patients with
PCR-negative and culture-PCR-negative results (i.e., 62 of 410 [15%])
than from the combined group of patients with PCR-positive
or culture-positive results (i.e., 4 of 102 [4%]) (P
,
0.01).
Since erythromycin inhibits pharyngeal colonization with B.
pertussis (5), we determined the frequency of patients receiving
erythromycin at the time that specimens were obtained for
culture. Eight of the 25 PCR-positive, culture-negative patients
were receiving erythromycin, whereas only 2 of 77
culture-positive patients were receiving erythromycin (P
,
0.001).
Other differences between the group of patients with
ture-confirmed pertussis and the group with PCR-positive,
ture-negative results were that more of the PCR-positive,
cul-ture-negative patients than the culture-positive patients had
received at least three doses of pertussis vaccine (10 of 23
versus 16 of 76; P
5
0.05) and that as a group the
PCR-positive, negative patients were older than the
culture-positive patients (13 of 25 PCR-culture-positive, culture-negative
pa-tients were 2 years of age or older, whereas 14 of 82
culture-positive patients were 2 years of age or older; P
,
0.01). Also,
a higher proportion of PCR-positive, culture-negative patients
than culture-positive patients coughed for at least 1 month (7
of 24 versus 2 of 74; P
,
0.01).
DISCUSSION
[image:3.612.96.258.69.177.2]Our multiplex PCR-based assay readily identifies
nasopha-ryngeal swab specimens that contain inhibitors of the PCR (28,
30). The amplification control system uses as a template for
b
-actin gene primers DNA from a human cell culture line
FIG. 2. Autoradiograph demonstrating positive (A), negative (B and C), andnonevaluable (D) results obtained from the testing of selected clinical specimens by the multiplex PCR-based assay. Specimens: A, companion swab culture pos-itive for B. pertussis; B to D, companion swabs culture negative for B. pertussis. W, reagent control.
TABLE 1. Comparison of the sensitivities and specificities of the
multiplex PCR-based assay and culture following the resolution of
discrepant results
aTest and result
No. of specimens
resolved as: Sensitivity (%)
Specificity (%) Positive Negative
PCR-based assay
Positive
93
1
98.9
99.7
Negative
1
394
Culture
Positive
69
0
73.4
100
Negative
25
395
a
Includes 489 of the 496 original pairs, as explained in the Results section.
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[image:3.612.316.556.100.206.2]rather than a plasmid construct containing B. pertussis primer
binding sites (27). It is therefore easier to prepare, and it can
also be incorporated into PCR-based assays for the detection
of other infectious agents. Only a small dilution (e.g.,
#
1 to 8)
of the specimen lysate is generally needed to overcome
inhi-bition, which was observed in 6% of the specimen.
Problems with carryover contamination are greatly
mini-mized with the multiplex PCR-based assay, since a positive
result requires amplification of two separate B. pertussis DNA
targets and hybridization with specific internal DNA probes.
This feature, the use of uracil-N
9
-glycosylase, and the physical
separation of specimen processing, PCR setup, and PCR
am-plification activities (24) appear to be effective control
mea-sures since we identified only one potentially false-positive
PCR result.
The multiplex PCR-based assay has high degrees of
analyt-ical sensitivity and specificity. It is capable of detecting nearly
a single input copy of B. pertussis DNA among excess human
DNA. This lower limit of sensitivity is made possible with the
detection of the PCR products by solution hybridization with
specific
32P-labeled internal DNA probes and autoradiography
(24, 29). Our findings that DNAs from a wide variety of
bac-teria isolated from the upper respiratory tracts of children, as
well as from strains of B. parapertussis and B. bronchiseptica, do
not yield PCR products that hybridize with the internal probes
agrees with earlier studies (2, 9, 23, 27).
For the detection of B. pertussis in nasopharyngeal swab
specimens, the multiplex PCR-based assay is considerably
more sensitive than the culture technique used in our study
(98.9 versus 73.4%, respectively). In an effort to optimize our
culture technique, specimens were inoculated at the side of the
patient onto a set of freshly prepared, selective, and
nonselec-tive Bordet-Gengou agar plates. However, the type of culture
medium (i.e., Bordet-Gengou agar versus Regan-Lowe
me-dium) may also affect the isolation rate of B. pertussis from the
nasopharynx. Some authorities (21) indicate that freshly
pre-pared Bordet-Gengou agar is equivalent to Regan-Lowe
me-dium for the isolation of B. pertussis. However, Ahmad and
Calder (1) reported a slightly higher isolation rate with
char-coal agar medium compared with that with Bordet-Gengou
agar and an additional improvement with the simultaneous use
of both media. The culture method can therefore greatly affect
the determination of the sensitivity of the PCR-based assay.
We chose to resolve PCR-positive, culture-negative
discrep-ant results by testing an additional aliquot of specimen
suspen-sions using the multiplex PCR-based assay rather than using
primers directed against a region of B. pertussis DNA other
than the reiterated sequence. Other PCR-based assays for B.
pertussis amplify single-copy targets (e.g., pertussis toxin and
adenylate cyclase toxin genes and DNA upstream from the
porin gene [19]) and are likely less sensitive than those based
on the repetitive element (9). For example, we found that a
PCR-based assay with primers for the pertussis toxin gene and
a specific internal probe (14) was 100-fold less sensitive than
the multiplex PCR-based assay when purified B. pertussis DNA
was tested. The toxin PCR-based assay was also used to
eval-uate selected specimens from patients that yielded various
densities (4) of B. pertussis on culture plates and a multiplex
PCR-positive result. The toxin PCR-based assay was positive
for only 5 of 10 specimens from patients with 1
1
to 2
1
growth
and for 9 of 10 specimens from patients with 3
1
to 4
1
growth
(P
5
0.07; Fisher exact test). We also found that samples from
only 9 of the 25 (36%) patients with multiplex PCR-positive,
culture-negative results yielded a positive result by the toxin
PCR-based assay. Therefore, although confirmatory testing of
the discrepant results with a primer pair for a different target
region (15) is helpful when the assays are of equal sensitivity,
this approach is not useful when the confirmatory assay is less
sensitive than the test under consideration. Our approach for
resolving PCR-positive, culture-negative results is also
consis-tent with the view of Lozano et al. (17) that repeatedly positive
PCR results for the detection of Junı´n virus should be
consid-ered equivalent to isolation of the virus.
Several consistencies in the results of the study also support
the view that the 25 patients with PCR-positive,
culture-nega-tive results represent patients with true cases of pertussis. First,
these cases of disease were temporally related to the cases of
culture-confirmed pertussis. If there had been clustering of
PCR-positive, culture-negative results during periods when the
rate of culture-confirmed pertussis was low or absent, they
would likely represent false-positive results of the PCR-based
assay. Second, the rates of documented viral infection in the
two groups were only 4%, compared with 15% in the group of
patients who had no evidence of pertussis infection. If the
group of patients with PCR-positive, culture-negative results
represent patients with false-positive results by the PCR-based
assay, we would expect similar rates of viral infection in this
group and the group without any evidence of pertussis
infec-tion. Third, review of historical information, which was
avail-able for 24 of the 25 patients, showed that 20 patients met the
Centers for Disease Control and Prevention (CDC) definition
of pertussis (cough lasting 14 days or longer with one or more
of the following: paroxysms of coughing, posttussive emesis, or
inspiratory whoop) (6). Of the four patients who did not satisfy
the CDC definition, two were receiving erythromycin, one was
the mother of one child who met the CDC definition, and the
other had a sibling who was culture positive. The single patient
for whom sufficient information was not available to apply the
CDC definition had a positive result by the pertussis toxin
PCR-based assay.
Our results suggest that PCR is particularly valuable in
de-tecting B. pertussis in the later stages of pertussis, in older
patients, in patients who have received pertussis immunization,
and in patients receiving erythromycin. In these situations the
density of B. pertussis in the nasopharynx is usually very low
and culture techniques are insensitive (3, 5, 12, 25, 31).
Treat-ment with erythromycin and other antibiotics may interfere
with the growth and therefore culture of B. pertussis (3, 5),
while PCR-based assays can detect the DNAs of both the
viable and nonviable bacteria. The presence of a low level of
antibody in the nasopharynx, enough to slow growth but not
enough to prevent infection with B. pertussis, might explain the
culture-negative but PCR-positive condition observed in
im-munized patients, older patients, and patients in the later
stages of infection, but other growth inhibitors in the
naso-pharynx could also be responsible.
Taking into account a small average batch size (e.g., five
specimens), controls, and repeated tests, we estimated for the
multiplex PCR-based assay a materials cost of $22.70 per
spec-imen and a technical time of 1.4 h per specspec-imen. Because the
assay is relatively expensive and technically demanding, it is
mainly recommended for use in specialized laboratories.
ACKNOWLEDGMENTS
This work was supported in part by the Pathology Education and
Research Foundation.
We thank John P. Anhalt for critical review of the manuscript and
Eduardo Yunis and Ellen R. Wald for encouragement.
REFERENCES
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