Optimizing polymerase chain reaction testing for the diagnosis of pertussis: current perspectives

Sophie Arbefeville, Patricia Ferrieri Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN, USA Abstract: Nucleic acid testing has revolutionized the diagnosis of pertussis in the clinical microbiology laboratory and has become the main avenue of testing for pertussis infection. Real-time polymerase chain reaction (RT-PCR) is an important tool for timely diagnosis of pertussis and is more sensitive than culture. The most commonly amplified targets are the insertion-sequence (IS) genes, which are found in multiple copies in the genome of Bordetella species. Some strains of Bordetella pertussis have more than 200 copies of IS481 in their genome. This high number of repeats allows RT-PCR assays to be very sensitive and makes nucleic acid testing two to three times more sensitive than culture. Despite these advantages, RT-PCR can give inaccurate results due to contamination or lack of specificity. Contamination can easily happen during specimen collection, DNA extraction, or nucleic acid amplification steps. To avoid contamination, laboratories need to have quality controls and good workflows in place. The poor specificity of the nucleic acid assays amplifying the IS genes is because they are found in various Bordetella species and, thus, not unique to a specific species. Bordetella holmesii, a more recently described Bordetella species found to be responsible for respiratory symptoms similar to pertussis in adolescents and adults, can be misidentified as B. pertussis in RT-PCR assays that amplify only the IS481 target. Use of multiple targets may improve specificity of RT-PCR assays for pertussis. In the past few years, the US Food and Drug Administration has cleared three commercial assays for the detection of B. pertussis in respiratory specimens. Several commercial assays and analyte-specific reagents, which are not US Food and Drug Administration cleared, are available for the detection of one or more Bordetella species by nucleic acid testing. Because of the diversity of nucleic acid amplification assays used, pertussis testing is not standardized across clinical laboratories. Keywords: nucleic acid testing, real-time PCR, Bordetella pertussis, Bordetella specie

Comparison of BD Phoenix AP Workflow with Vitek 2▿

The BD Phoenix AP instrument reduced the manual setup time for the Phoenix system by 50%. For batches of 14 organisms, the average manual manipulation time per isolate was 89.5 s for BD Phoenix by the use of the AP instrument and 101 s for Vitek 2 (P < 0.001)

Epidemic 2014 enterovirus D68 cross-reacts with human rhinovirus on a respiratory molecular diagnostic platform.

Enterovirus D68 (EV-D68) is an emerging virus known to cause sporadic disease and occasional epidemics of severe lower respiratory tract infection. However, the true prevalence of infection with EV-D68 is unknown, due in part to the lack of a rapid and specific nucleic acid amplification test as well as the infrequency with which respiratory samples are analyzed by enterovirus surveillance programs. During the 2014 EV-D68 epidemic in the United States, we noted an increased frequency of "low-positive" results for human rhinovirus (HRV) detected in respiratory tract samples using the GenMark Diagnostics eSensor respiratory viral panel, a multiplex PCR assay able to detect 14 known respiratory viruses but not enteroviruses. We simultaneously noted markedly increased admissions to our Pediatric Intensive Care Unit for severe lower respiratory tract infections in patients both with and without a history of reactive airway disease. Accordingly, we hypothesized that these "low-positive" RVP results were due to EV-D68 rather than rhinovirus infection. Sequencing of the picornavirus 5' untranslated region (5'-UTR) of 49 samples positive for HRV by the GenMark RVP revealed that 33 (67.3%) were in fact EV-D68. Notably, the mean intensity of the HRV RVP result was significantly lower in the sequence-identified EV-D68 samples (20.3 nA) compared to HRV (129.7 nA). Using a cut-off of 40 nA for the differentiation of EV-D68 from HRV resulted in 94% sensitivity and 88% specificity. The robust diagnostic characteristics of our data suggest that the cross-reactivity of EV-D68 and HRV on the GenMark Diagnostics eSensor RVP platform may be an important factor to consider in making accurate molecular diagnosis of EV-D68 at institutions utilizing this system or other molecular respiratory platforms that may also cross-react

PCR amplification of a portion of the 5’-UTR from GenMark eSensor RVP samples.

<p>cDNA synthesized from RNA extracted from de-identified and masked RVP samples was used for traditional PCR amplification of the 5’-UTR. Primers were described by Oberste, <i>et</i>. <i>al</i>., for the sequencing of EV-D68 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118529#pone.0118529.ref002" target="_blank">2</a>]. Agarose-ethidium bromide electrophoresis demonstrated that 49 of 62 samples analyzed yielded a 396 base pair amplicon compatible with EV-D68. Results shown corresponded to samples 2–4, 6–13, and 15–17, and are representative of the range of band intensities observed for all positive samples. NTC: no template control.</p