SUMMARYRespiratory viral infections are associated with a wide range of acute syndromes and infectious disease processes in children and adults worldwide. Many viruses are implicated in these infections, and these viruses are spread largely via respiratory means between humans but also occasionally from animals to humans. This article is an American Society for Microbiology (ASM)-sponsored Practical Guidance for Clinical Microbiology (PGCM) document identifying best practices for diagnosis and characterization of viruses that cause acute respiratory infections and replaces the most recent prior version of the ASM-sponsored Cumitech 21 document,Laboratory Diagnosis of Viral Respiratory Disease, published in 1986. The scope of the original document was quite broad, with an emphasis on clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen detection and viral culture. The new PGCM document is designed to be used by laboratorians in a wide variety of diagnostic and public health microbiology/virology laboratory settings worldwide. The article provides guidance to a rapidly changing field of diagnostics and outlines the epidemiology and clinical impact of acute respiratory viral infections, including preferred methods of specimen collection and current methods for diagnosis and characterization of viral pathogens causing acute respiratory tract infections. Compared to the case in 1986, molecular techniques are now the preferred diagnostic approaches for the detection of acute respiratory viruses, and they allow for automation, high-throughput workflows, and near-patient testing. These changes require quality assurance programs to prevent laboratory contamination as well as strong preanalytical screening approaches to utilize laboratory resources appropriately. Appropriate guidance from laboratorians to stakeholders will allow for appropriate specimen collection, as well as correct test ordering that will quickly identify highly transmissible emerging pathogens.

Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported.Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.Bordetella pertussis is the causative agent of whooping cough, an infectious disease that occurs worldwide with a high prevalence among young, unvaccinated infants (2,3,6,19) and is recently resurgent in highly vaccinated populations. Related species, including B. parapertussis, B. bronchiseptica, and the more recently described B. holmesii (27), may also cause a pertussis-like syndrome in humans. Laboratory diagnosis of pertussis is traditionally based primarily on culture, which is highly specific but is maximally sensitive only in the initial phases of disease (10,14). Furthermore, culturing depends on specimen quality and laboratory expertise and requires special media, extended incubation periods of 7 days or more, and confirmation by biochemical or antibody reagent tests. Diagnostic serology can be highly sensitive and more rapid than culture (14), but no serologic assay has been approved for diagnostic use in the United States because no diagnostic criterion has been widely accepted and no method has been validated between laboratories.Thus, there is a need for more rapid and sensitive diagnostic methods that have high positive predictive value, especially in the early stages of disease. As is the case for other fastidious organisms, PCR offers an attractive alternative for detecting B. pertussis and B. parapertussis in clinical specimens (1,4,5,7,15,17,21,26). Previously evaluated B. pertussis target regions include insertion sequences (IS), repeat elements, the pertussis toxin promoter region, the adenylate cyclase gene, and the porin gene. IS481 is present in the B. pertussis genome at 80 (22) to 100 (5, 18) copies, and PCR assays targeting IS481 have been evaluated for sensitivity and specificity in several laboratories over the last few years. Therefore, an internal region of IS481 was selected as the B. pertussis target (18) while a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) duplex PCR for B. pertussis and B. parapertussis was being developed. The previously described (5) forward primer BP-1 (5Ј GAT TCA ATA GGT TGT ATG CAT GGT T 3Ј and the slightly modified reverse primer BP-2 (5Ј TTC AGG CAC ACA AAC TTG ATG GGC G 3Ј), corresponding to bp 12 to 36 and 192 to 167 (GenBank M22031) of IS481, respectively, were used for amplification. A pair of fluorescence-labeled hybridization probes, BP-HP-3 (5Ј TCG CCA ACC CCC CAG TTC ACT CA-FAM 3Ј) and BP-HP-4 (5Ј LC red 640-AGC CCG GCC GGA TGA ACA CCC-3Ј-phosphate), corresponding to bp 66 to 88 and 92 to 112 (GenBank M22031) of IS481, respecti...

We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis.

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.

Several studies have demonstrated that the sensitivity of a commercially available PCR test for the detection of Chlamydia trachomatis (Roche Diagnostics) is affected by the cellular quality of the endocervical swab specimens. The cellular adequacies of 1,633 female endocervical swab specimens were assessed and compared with the results of C. trachomatis detection obtained by ligase chain reaction (LCR; Abbott Laboratories). Specimen adequacy studies and LCR were performed with samples from the same swab, after demonstration of the stability of human epithelial cells in LCR transport medium. Prior to heat treatment of the swab specimen, an aliquot was removed and cytocentrifuged onto a slide. Cell spots were stained and examined at ؋400 magnification for endocervical (columnar epithelial or metaplastic) cells and erythrocytes. The overall rate of positivity of the LCR was 6.5% (106 of 1,633 specimens) with pooled specimens (pools of 4 specimens each; reduced cutoff). Of the 1,633 specimens examined, 655 (40.1%) were found to contain one or more endocervical cells. The rate of positivity for C. trachomatis was 10.8% (71 of 655 specimens) among specimens containing endocervical cells, whereas it was 3.6% (35 of 978 specimens) among specimens lacking endocervical cells (P < 0.0001). There was no linear trend between the rate of positivity for C. trachomatis and the number of endocervical cells (P ‫؍‬ 0.24). The rate of positivity for C. trachomatis was 5.4% (8 of 147 specimens) among specimens containing large numbers of erythrocytes (>100 per high-power field), whereas it was 6.6% (98 of 1,486 specimens) among specimens containing less than 100 erythrocytes per high-power field (P ‫؍‬ 0.59). These results show that the sensitivity of the Abbott C. trachomatis LCR test is affected by the presence of endocervical cells. Additionally, they indicate that the presence of a single endocervical cell is as good an indicator of specimen adequacy as the presence of many endocervical cells. The presence of a large number of erythrocytes was not associated with an increased rate of sensitivity of the LCR.Chlamydia trachomatis is the most common bacterial sexually transmitted organism in the United States, with an estimated annual incidence of infections with this organism of 3 million to 4 million (3, 4). A variety of commercially available test methods exist for the detection of C. trachomatis infections, including cell culture, antigen detection enzyme immunoassay, direct fluorescent-antibody assay, nonamplified nucleic acid hybridization tests, and nucleic acid amplification tests. The analytical and clinical sensitivities of these methods vary greatly (2). In addition, the sensitivities of direct fluorescent-antibody assay, enzyme immunoassay, and nucleic acid hybridization tests have been shown to be affected by the cellular adequacy (presence of columnar epithelial or metaplastic [endocervical] cells or large numbers of erythrocytes [more than 100 per high-power microscopic field]) of female endocervical swab specimens ...