hIS1001, pIS1001, and ptxS1 targets. The PCR results of these nine specimens were interpreted as indeterminate. High occurrence (18.6%) of indeterminate PCR results in Bordetella PCR testing has been reported previously (16). Specimens with indeterminate PCR results can be true positive with low bacterial loads or false positive because of contamination of B. pertussis from patient clin- ics or the laboratory (11). Most of the specimens in this study, from both pertussis cases and noncases, were collected within 2 weeks after onset and stored and transported cold; therefore, the negative or indeterminate PCR results do not appear to be related to poor specimen quality or specimen management (17). Addi- tionally, in most cases, a dilution step and repeat testing of speci- mens that were RNP negative did not change the overall result, indicating that inhibition may not be a significant concern in this assay. Among the nine individuals with indeterminate PCR re- sults, four had pertussis and five did not. Samples from two of the four pertussis cases with indeterminate PCR results tested positive by culture. This reinforces the usefulness of culture for the iden- tification of Bordetellaspecies in specimens with high IS481 C T
Blood sample and specimen Collection. Blood samples of sheep and poultry were collected from local slaughter houses and the local market Nanded, Maharashtra under the supervision of veterinary practitioners, while human blood samples were obtained from normal healthy volunteers by lab technicians in sterile bulbs. Bordetellaspecies were isolated from the specimen (n=50) collected from nasopharynx region of suspected whooping cough patients using calcium alginate swabs during the period of April 2006 to April 2007. The swabs were immediately stored in Stainer Scholte transport media to maintain cell viability and used without delay. All the fifty samples were collected and processed for bacterial isolation.
While PCR is the most common method used for detecting Bordetella pertussis in the United States, most laboratories use inser- tion sequence 481 (IS481), which is not specific for B. pertussis; therefore, the relative contribution of other Bordetellaspecies is not understood. The objectives of this study were to evaluate the proportion of other Bordetellaspecies misidentified as B. per- tussis during a period of increased pertussis incidence, determine the level of agreement in Bordetellaspecies detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDC’s PCR assay when using a differ- ent PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories for B. pertussis and B. parapertussis detection. Every fifth specimen positive for IS481 and/or IS1001 with cycle threshold (C T ) values of
Coinfections of B. parapertussis and B. pertussis have been documented (14). In our clinical specimens, two coinfections of B. pertussis and B. parapertussis were potentially identified, with IS481 C T values of less than 35 and pIS1001-positive results suggesting that the specimens were most probably coin- fections of the two species. These results exemplify the robust- ness of the multiplex assay. Although we identified one coin- fection of B. holmesii and B. pertussis (IS481, hIS1001, and ptxS1 positive and pIS1001 negative) using our PCR multitar- get assay, it was not culture confirmed. However, we recently identified a coinfection of B. holmesii and B. pertussis by cul- ture (unpublished data). For coinfections of B. pertussis and B. parapertussis or B. holmesii, our RT-PCR data are very limited; however, C T values for the appropriate targets of less than 40 with a ptxS1 assay-positive result are suggestive of coinfections. The results from testing of 402 characterized clinical isolates substantiate the value of our algorithm to discriminate among Bordetellaspecies. Since our pIS1001 primers/probe cross-re- acted with 4 human-derived B. bronchiseptica isolates (5.5%), all B. bronchiseptica isolates may not be distinguished from B. parapertussis by this algorithm. In B. bronchiseptica, the pres- ence of IS481 is host dependent (20), and human isolates of B. bronchiseptica may contain IS481 as well (17). Using our prim- ers, we found only 1 of 73 human B. bronchiseptica isolates (strain BBE001, isolated in 1956) with IS481 homology, but the amplification results were atypical and none of the more recent human isolates in our collection had IS481 detectable by our RT-PCR assay. Genome sequencing of two human-derived B. bronchiseptica isolates, BBE001 and BBF579 (isolated in 2007), was performed (13). These results confirmed the IS481 sequences (Fig. 1) for BBE001 and demonstrated that BBF579 did not contain IS481 sequences.
anti-PT antibodies are highly specific for B. pertussis infection (18, 20). Anti-FHA antibodies are less specific than PT anti- bodies and may be produced following infection with Myco- plasma pneumoniae, Chlamydia pneumoniae, nonencapsulated Haemophilus influenzae, or other Bordetellaspecies, such as B. parapertussis and B. bronchiseptica (7, 16, 18). Adenylate cy- clase toxin (ACT) antibodies have also been proposed to be a sensitive marker for Bordetella sp. infection (27). The detection of IgA antibodies against PT and FHA suggests natural infec- tion not immunization, since IgA is rarely if at all detected following immunization (17, 21). However, the detection of IgG to PT and FHA is more sensitive than that of IgA, since not all individuals mount a detectable IgA response following infection (22). An IgM response is only rarely and inconsis- tently seen following exposure or immunization (25). Addition- ally, since both PT and FHA are included in the acellular pertussis vaccines, antibodies to these antigens often persist fol- lowing vaccination, hindering serodiagnosis in vaccinated individ- uals. Therefore, standardization of the diagnostic threshold would be important for the detection of active infection.
The Aries Bordetella assay (Aries BA) uses Luminex Corporation’s MultiCode real-time PCR chemistry in combination with the Aries systems. The system is capable of automated nucleic acid extraction and puriﬁcation, real-time PCR detection of nucleic acid sequences, and data analysis. The Aries Bordetella assay can directly detect and differentiate B. pertussis and B. parapertussis from nucleic acid in nasopharyngeal swab (NPS) samples from patients with signs and symptoms of whooping cough. The Aries Bordetella assay targets the pertussis toxin (ptxA) promoter and IS1001 repeat sequence in the genomes of B. pertussis and B. parapertussis, respectively. The promoter toxin gene targeted by the Aries Bordetella assay confers a high degree of speciﬁcity and is designed to detect only B. pertussis, unlike other PCR-based assays, such as those that target the IS481 region. In contrast, IS481, a multicopy insertion element, is present in multiple Bordetellaspecies, including B. pertussis, Bordetella holmesii, and Bordetella bronchiseptica (7–9).
PCR results with clinical samples. The results obtained by various investigators with different PCR systems performed with clinical samples are presented in Table 1. All PCR meth- ods proved to be very specific, although some systems could not distinguish B. pertussis from other Bordetellaspecies. When nasopharyngeal samples were analyzed by culture and PCR, PCR was found to be significantly more sensitive than culture overall (1,462 versus 931 samples; P , 0.001, chi-square test), although the samples in different studies were collected from different patient populations. PCR identified more pertussis patients than culture in each study. Only a minority of samples (2%) were PCR negative and culture positive, and all PCR systems were able to score 80 to 100% of culture-positive samples as PCR positive. However, there were large differ- ences in the percentage of PCR-positive samples that were culture negative (2 to 51%). These differences may have been due to differences in study populations (vaccination status, antibiotic pretreatment, stage of disease, etc.), clinical samples (type, source), culture procedures, and the sensitivities of dif- ferent detection systems. The interpretation of PCR-positive but culture-negative samples as either true positive or false positive is difficult. To confirm the result, PCR should be re- peated or the result should be confirmed by another method (DFA testing or serology) (20, 24, 30, 91). A culture-negative but PCR-positive result should be considered a true-positive result only when the patient has symptoms typical of those of pertussis according to WHO criteria for clinical pertussis (91). A PCR-positive but culture-negative result is common at later stages of the disease, in vaccinated patients, and in patients who are under antibiotic treatment or who have recently had close contact with patients with culture-proven pertussis (24). The significance of results of PCR with clinical specimens from two large clinical populations with high levels of pertussis vaccination coverage has been analyzed (41). In Finland, of a total of 1,904 clinical samples tested for pertussis by PCR, 447 (23%) were positive. By culture and PCR a total of 829 sam- ples were tested, and an additional 7 samples were only culture positive. The PCR positivity rate correlated with the number of B. pertussis colonies on plates. In Switzerland, 1,830 routine clinical samples were tested for B. pertussis by PCR only. A total of 683 were PCR positive, and 29 of 868 control samples were PCR positive. The comparison of culture, PCR, and en- zyme immunoassay results demonstrated that none of the methods was able to detect all B. pertussis infections: 25% were confirmed by culture, 61% were confirmed by PCR, and 57% were confirmed by enzyme immunoassay.
Global searches in Bordetellaspecies have identiﬁed numerous BvgA-regulated genes. Microarray analyses identiﬁed ~250 genes regulated in B. pertussis Tohama I BP536 and nearly 500 genes regulated in B. bronchiseptica RB50 (9, 31). Known vags, including those encoding adhesins and toxins, were expressed consistently in both species in the Bvg( ⫹ ) mode, while more species-speciﬁc differences were observed in the Bvg( ⫺ ) mode. Recently, Ahuja et al. reported the ﬁrst transcriptome sequencing (RNA-seq) analysis in a Bordetellaspecies, but it was limited to B. bronchiseptica (32). Here, we report the ﬁrst RNA-seq examination of the BvgAS regulon in B. pertussis. Our work identiﬁes more than 550 genes within the BvgAS regulon with 245 transcripts positively regulated and 326 transcripts negatively regulated by BvgA~P. Our work suggests that B. pertussis has hierarchical regulatory networks under the BvgA phos- phorelay. Positively regulated genes include many transcriptional regulators and genes within the type 3 secretion system (T3SS). We demonstrate that expression of the extracytoplasmic function (ECF) sigma factor brpL, which is needed for T3SS expression and is signiﬁcantly upregulated in the Bvg( ⫹ ) mode, is directly activated by BvgA~P. In addition, we ﬁnd that multiple genes involved in metabolic pathways are negatively regulated by BvgAS and thus upregulated in the Bvg( ⫺ ) mode. Our results are consistent with the idea that metabolic changes in the Bvg( ⫺ ) mode may be partici- pating in bacterial survival, transmission, and/or persistence.
Biochemical tests that differentiate NO-2 from Bordetellaspecies and other phenotypically similar bacteria are listed in Table 4. The lack of oxidase activity and the production of a brown soluble pigment differentiate NO-2 from B. pertussis, B. bronchiseptica, and B. avium. The lack of urease activity differentiates NO-2 from B. parapertussis. Other phenotypically similar bacteria include asaccharolytic Acinetobacter species and CDC group NO-1 (15). Like NO-2, these organisms are usually oxidase negative and do not utilize carbohydrates; however, they usually do not produce a brown soluble pigment, and group NO-1 reduces nitrate. Rarely occurring asaccharo- lytic Acinetobacter species that produce a brown soluble pig- ment can be differentiated from NO-2 by the Acinetobacter transformation assay and CFA analysis (4, 31). Brucella canis TABLE 3. CFA composition of CDC group NO-2, B. avium,
The lower B. pertussis sensitivities achieved for some samples by laboratories that extract DNA with MagNA Pure or manual Qiagen spin columns suggest that these two methods may be less efficient at DNA extraction than the EasyMAG platform or simple heat lysis. A disadvantage of using heat lysis to release DNA for PCR is that it does not remove potential PCR inhibitors. Although human DNA was added to the bacterial saline suspensions to sim- ulate clinical specimens, actual specimens may contain inhibiting components. An additional concern with manual extraction or heat lysis is the increased possibility for DNA cross-contamina- tion (15). An efficacy comparison of DNA extraction kits to detect Bordetellaspecies in nasopharyngeal swabs or aspirates without the added complication of multiple laboratories and PCR proto- cols will be required to definitively resolve this question.
In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramag- netic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetellaspecies (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR- based assay, while the culture assay was negative. The implications of these results are discussed.
The present study showed that PCR, using the primers PIp1 and PIp2, was specific for B. pertussis with one exception: B. holmesii gave rise to a band indistinguishable from that in- duced by B. pertussis. This band also hybridized to the PI probe. The risk of B. holmesii giving false-positive results by pertussis PCR is minimal because the organism has not been found in the respiratory tract and does not cause a disease similar to pertussis. The organism has been isolated from the blood of a few septic patients (12, 25). Bacterial suspensions of the other 5 Bordetellaspecies and of another 13 bacterial spe- cies that can be found in the respiratory tract gave no false- positive reactions.
the PvuII ribotype of MBORD669 is unique among our IS481- positive isolates (40; K. B. Register, unpublished data). A com- parative evaluation of MBORD669 DNA obtained from each laboratory could perhaps assist in resolving the discrepancy. The remaining study (50), in which the method used to identify IS481 was not detailed, included 76 strains examined here. Our results are in agreement with theirs, except for five isolates reported here as positive for IS481 which were previously re- ported as IS481 negative. In the absence of additional experi- mental details, the reason for this discrepancy is unclear. It could be postulated that our results are confounded by the presence of IS1002, an insertion sequence closely related to IS481 found in multiple Bordetellaspecies including, rarely, B. bronchiseptica (8, 49). However, the degree of sequence iden- tity between IS1002 (49) and our IS481 probe is insufficient to withstand the stringency of the conditions used for hybridiza- tions. Detection of many identically sized restriction fragments with the IS481 probe in all strains positive by Southern blotting (Fig. 3A; K. B. Register, unpublished data) also argues against cross-hybridization of the probe with IS1002.
The clinical significance of detecting B. holmesii is not yet fully understood, but the bacterium has been isolated from the respiratory tract (16, 35) and been reported to cause disease (24). However, because of the design of the PCR assays de- scribed it is possible to provide a presumptive diagnosis for B. holmesii, as both assays detect B. holmesii. However, a mixed infection of B. pertussis and B. parapertussis is also possible but rare, and to date only one has been reported (11). Essentially any PCR assay targeting the IS481 region will detect B. pertus- sis and B. holmesii (22), so the use of the B. parapertussis PCR, which detects both B. holmesii and B. parapertussis, enables B. holmesii infection or a dual infection of B. pertussis and B. parapertussis to be diagnosed. In the rare event that this occurs, the PCR product from the B. parapertussis PCR can be sequenced and B. holmesii infection can be distinguished from a dual infection. Of other Bordetellaspecies such as B. bron- chiseptica, B. avium, B. trematum, and B. hinzii, it is only B. bronchiseptica that has been reported to cause respiratory infection in immunocompromised hosts (14). So the possibility
Real-time PCR formats have increased the speed and sen- sitivity of diagnosis compared to culture, although all proce- dures need more standardization and optimization. Recom- mendations include extraction controls, internal controls, a B. pertussis and B. parapertussis DNA standard, and participation in an external QC program. For epidemiology and surveillance purposes, the role of B. holmesii and of other Bordetellaspecies may require a genus-specific real-time PCR format, followed by a species-specific format. So far, no published procedure has comprised all necessary controls including those for consis- tency of bacterial lysis and yield of DNA extraction. Commer- cially available kits with appropriate validation would signifi- cantly facilitate the work of medical laboratories, and such CE-marked kits are a requirement in the European Union as from 2005. Further developments in automation of sample preparation and PCR procedures are ongoing, and Bordetella PCR will be just one of many applications to be run on mech- anized PCR systems.
A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopha- ryngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC- PCR-IS assay for a different group of nasopharyngeal swab specimens (n ⴝ 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P ⴝ 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.
The regions of IS481 and BP0026, targeted in our assay are sensitive with LLOD of 1 and 10 B. per- tussis genomic equivalent per reaction, respectively. The region of IS1001 targeted in our assay is sensitive with LLOD of 1 B. parapertussis genomic equivalent per reaction. All regions are specific with no cross- reactivity with non-Bordetella spp. or human DNA. It is known that a small amount of IS481 copies are found in the genomes closely related with pathogens B. bronchiseptica and B. holmesii (27,37). In order to identify DNA of these pathogens in our real-time PCR design of primers and probe for selection IS481 was performed on the fragment with 99% homology to the nucleotide sequences IS481 from B. bronchi- septica (accession no. EF043395) and B. holmesii (accession no. DQ420073). Unfortunately two avail- able B. bronchiseptica strains were negative in our real-time PCR and there were no strains of B. holme-
sheep and turkey erythrocytes by B. avium and B. hinzii and found that the outcome is affected by the method used (Reg- ister, unpublished data). Most B. hinzii isolates are weakly positive when a 96-well plate procedure is employed (18) but negative when tested by a slide agglutination method (22). Currently, there is no readily available technique for reliably distinguishing between B. avium and B. hinzii. The results reported here demonstrate that both ribotyping and REA are equally useful for this purpose. Ribotyping may be preferable since a quick visual examination of the resulting patterns is the only postassay analysis needed. In contrast, the complexity of REA patterns requires computer-assisted analysis for interpre- tation. 16S rRNA gene sequencing has recently been proposed as a method for definitive identification of B. hinzii (14). Only a single B. avium isolate was included in that study, but the numerous base pair differences found between B. avium and B. hinzii suggest this technique would also detect other B. avium isolates, although this has yet to be demonstrated. While 16S rRNA sequencing is a high-resolution technique, it requires amplification of genomic DNA and takes considerably longer to complete than ribotyping. No PvuII sites are present in the B. avium and B. hinzii 16S rRNA sequences reported, indicat- ing that the sequence variability responsible for distinguishing these species by ribotyping must be located in other regions of the rrnB operon included in the probe.