A simplified sequence-based identification scheme for Bordetella reveals several putative novel species.

The differentiation of Bordetella species, particularly those causing human infection, is problematic. We found that sequence analysis of an internal fragment of nrdA allowed differentiation of the currently named Bordetella species. Analysis of 107 "Bordetella" isolates recovered almost exclusively from human respiratory tract specimens identified several putative novel species.

Occurrence of 3 Bordetella species during an outbreak of cough illness in Ohio: epidemiology, clinical features, laboratory findings and antimicrobial susceptibility.

BACKGROUND: An increase in laboratory diagnosis of pertussis was noted in central Ohio during 2010. Diagnosis was made using a polymerase chain reaction assay targeting the multicopy insertion sequence IS481, which is found in both Bordetella pertussis (Bp) and Bordetella holmesii (Bh). An increase in specimens testing positive for Bordetella parapertussis (Bpp) using insertion sequence IS1001 was also noted. METHODS: Nasopharyngeal swab specimens submitted April 1, 2010, to March 31, 2011, were tested using a multiplex polymerase chain reaction assay for Bp/Bh (IS481) and Bpp followed by singleplex assays for Bp and Bh. A subgroup of specimens was also cultured for Bordetella species, and antimicrobial susceptibility testing was performed on recovered organisms. Demographic and clinical features were compared for patients with Bp, Bh and Bpp. RESULTS: Of 520 IS481-positive specimens, 214 (41.1%) were positive for Bp, 79 (15.2%) were positive for Bh and 5 (1.0%) were positive for both Bp and Bh; 222 (42.7%) were negative for both targets. An additional 220 specimens were positive for Bpp. Among a sample of 155 IS481-positive specimens, 40, 15 and 0 were culture positive for Bp, Bh and Bpp, respectively. Among a sample of 55 BparaIS1001-positive (Bpp) specimens, 22, 0 and 0 were culture positive for Bpp, Bp and Bh, respectively. All Bordetella species were susceptible to macrolide antibiotics. Patients with Bh were older than patients with Bp, who were older than those positive for Bpp (mean ages: 12.0, 8.0 and 4.2 years, respectively; P < 0.001). One or more classic signs of pertussis (ie, paroxysmal cough, whoop, post-tussive emesis) were seen in 55.9% of 263 patients (59 Bp, 24 Bh, 80 Bpp and 100 negative for Bordetella species), but did not differ statistically among the groups (χ = 5.1, P = 0.17). CONCLUSIONS: All 3 Bordetella species, Bp, Bh and Bpp, were detected during on outbreak of pertussis-like cough illness. There were noted differences in age and seasonality, but clinical features at the time of presentation did not allow clear differentiation of these infections. All Bordetella species recovered from culture and tested were susceptible in vitro to macrolide antibiotics. Additional study is necessary to further characterize epidemiologic and clinical characteristics of Bh-associated cough illness and to determine potential co-occurrence of Bordetella species with other bacterial and viral respiratory tract pathogens.

Clinical evaluation of a real-time PCR assay for identification of Salmonella, Shigella, Campylobacter (Campylobacter jejuni and C. coli), and shiga toxin-producing Escherichia coli isolates in stool specimens.

Enteric illness affects millions of individuals annually in the United States and results in >50,000 hospitalizations. The rapid and accurate identification of bacterial pathogens associated with gastroenteritis can aid acute patient management decisions, including the use of antibiotic therapy and infection control. This study compared the ProGastro SSCS multiplex real-time PCR assay (Gen-Probe Prodesse, San Diego, CA) to culture for the identification of Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Salmonella spp., and Shigella spp. and to broth enrichment followed by an FDA-cleared enzyme immunoassay (EIA) for the identification of Shiga toxin-producing Escherichia coli (STEC) isolates in stool specimens. Stool samples submitted in preservatives for routine culture and EIA were prospectively enrolled and tested at four clinical centers. Discrepancies between the ProGastro SSCS assay and culture or EIA were resolved using bidirectional sequencing. The overall prevalence of the pathogens as detected by culture was 5.6% (1.8% Campylobacter, 1.8% Salmonella, 1.3% Shigella, and 0.8% STEC). When results based on the ProGastro SSCS assay and bidirectional sequencing were applied, the overall prevalence increased to 8.3% (2.3% Campylobacter, 2.6% Salmonella, 1.8% Shigella, and 1.6% STEC). Following resolution of the discrepant results, the sensitivity of the ProGastro SSCS assay was 100% for all pathogens, and the specificities ranged from 99.4% to 100%. The sensitivity of culture compared to sequence-confirmed ProGastro SSCS results ranged from 52.9% to 76.9%, with the specificities ranging from 99.9% to 100%. Overall, these results suggest that the ProGastro SSCS assay is highly sensitive and specific in a clinical setting.

Purulent pericarditis secondary to community-acquired, methicillin-resistant Staphylococcus aureus in previously healthy children. A sign of the times?

RATIONALE: Purulent pericarditis secondary to community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) is a potentially lethal infection that has yet to be described in the pediatric population. Only four cases of purulent pericarditis secondary to CA-MRSA have been described in the English literature, all of whom were adults. OBJECTIVES: We report on the first two pediatric cases of purulent pericarditis secondary to CA-MRSA to increase awareness of this potentially fatal condition. METHODS: Clinical data were obtained from an 8-year-old male patient and a 7-month-old female patient, both previously healthy, who presented to our hospital for treatment of severe shock and multiorgan failure. Literature review was performed using MEDLINE and Cochrane databases. Pulsed-field gel electrophoresis was performed to confirm the organism type. MEASUREMENTS AND MAIN RESULTS: Our previously healthy patients presented with refractory shock and were found to have purulent pericarditis with tamponade secondary to CA-MRSA. Both patients required emergent pericardiocentesis and surgical pericardial debridement. Isolates from both patients were found to be MRSA USA type 300, a common type of CA-MRSA that has become the most frequent cause of skin and soft tissue infections in the United States. CONCLUSIONS: Purulent pericarditis survival hinges upon early empiric antibiotic therapy targeting resistant Staphylococcus, rapid diagnostic efforts, and expeditious pericardial drainage when diagnosed. An aggressive multidisciplinary approach provided for complete recovery in both cases, and both children were discharged with normal cardiac function. These two cases emphasize the need for consideration of CA-MRSA presenting with purulent pericarditis as an etiology for refractory shock.

Use of APTIMA Combo 2: the experience of a child advocacy center.

The Centers for Disease Control and Prevention recommends nucleic acid amplification testing for chlamydia and gonorrhea in sexually abused girls. No studies describe performance of APTIMA Combo 2 Assay with second target confirmation on the same testing platform. This nucleic acid amplification testing is evaluated within a large child advocacy center. Girls 3 to 18 years, 35% of whom reported consensual sexual activity, were prospectively tested by APTIMA Combo 2 on urine/vaginal swabs and by vaginal culture. A case of infection was defined as positive culture or positive urine or vaginal swab nucleic acid amplification testing with second target confirmation. Sensitivity of APTIMA Combo 2 on urine was found to be superior to vaginal culture and comparable to APTIMA Combo 2 on vaginal swabs for both infections. APTIMA Combo 2 on urine is less invasive, and its use may be preferred in this traumatized population.

Point: Should all stools be screened for Shiga toxin-producing Escherichia coli?

In October 2009, the Centers for Disease Control and Prevention recommended that clinical laboratories test all stools submitted for the detection of enteric bacterial pathogens for the presence of Shiga toxin-producing Escherichia coli (STEC). In order to do this, it is recommended that all stools be cultured for Escherichia coli O157:H7 on selective medium as well as that testing for the presence of Shiga toxin be done by immunoassay to detect non-O157 STEC (3). There are a variety of products that are FDA approved for detection of Shiga toxin. Further, it is recommended that Shiga toxin detection be done by testing overnight enrichment broth cultures of stools rather than directly examining stools for this toxin. This recommendation was made approximately 18 months ago. We have asked Mario Marcon of Nationwide's Children Hospital in Columbus, OH, to explain the rationale for his decision to follow this recommendation, while we have asked Deanna Kiska and Scott Riddell of Upstate University Hospital in Syracuse, NY, why these guidelines have not been adopted by their laboratory.

Comparison of polyurethane foam to nylon flocked swabs for collection of secretions from the anterior nares in performance of a rapid influenza virus antigen test in a pediatric emergency department.

Rapid antigen testing of upper respiratory secretions collected with various swab types is often utilized for laboratory diagnoses of influenza virus infection. There are limited data on the effects of swab composition on test performance. This study compared the performance of the Quidel QuickVue Influenza A+B test on secretions from the anterior nares when a polyurethane foam swab was used for collection to that when a nylon flocked swab was used for collection. One hundred subjects who presented to a pediatric emergency department with symptoms suggestive of an influenza virus infection were recruited for the study. Foam and flocked swabs of the anterior nares were obtained from separate nares of each subject before a posterior nasopharyngeal swab was collected and placed into viral transport medium. The QuickVue test was performed directly on each swab type, and the results were compared to the results of reverse transcription-PCR (RT-PCR), direct fluorescent antibody (DFA) test, and viral culture performed on the transport medium. RT-PCR alone and DFA combined with culture were utilized as separate gold standards. There were 56 cases of influenza detected by RT-PCR; the QuickVue test was positive for 40 foam and 30 flocked swabs, for sensitivities of 71% and 54%, respectively (P = 0.01). Similarly, there were 49 influenza cases detected by DFA and/or culture; the QuickVue test was positive for 38 foam and 30 flocked swabs, for sensitivities of 78% and 61%, respectively (P = 0.13). This study suggests that polyurethane foam swabs perform better than nylon flocked swabs for the collection of secretions from anterior nares in the Quidel QuickVue Influenza A+B test.

A one-step, real-time PCR assay for rapid detection of rhinovirus.

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

A decision rule for predicting bacterial meningitis in children with cerebrospinal fluid pleocytosis when gram stain is negative or unavailable.

OBJECTIVES: Among children with cerebrospinal fluid (CSF) pleocytosis, the task of separating aseptic from bacterial meningitis is hampered when the CSF Gram stain result is unavailable, delayed, or negative. In this study, the authors derive and validate a clinical decision rule for use in this setting. METHODS: This was a review of peripheral blood and CSF test results from 78 children (< 19 years) presenting to Children's Hospital Columbus from 1998 to 2002. For those with a CSF leukocyte count of > 7/microL, a rule was created for separating bacterial from viral meningitis that was based on routine laboratory tests, but excluded Gram stain. The rule was validated in 158 subjects seen at the same site (Columbus, 2002-2004) and in 871 subjects selected from a separate site (Boston, 1993-1999). RESULTS: One point each (maximum, 6 points) was assigned for leukocytes > 597/microL, neutrophils > 74%, glucose < 38 mg/dL, and protein > 97 mg/dL in CSF and for leukocytes > 17,000/mL and bands to neutrophils > 11% in peripheral blood. Areas under receiver-operator-characteristic curves (AROCs) for the resultant score were 0.98 for the derivation set and 0.90 and 0.97, respectively, for validation sets from Columbus and Boston. Sensitivity and specificity pairs for the Boston data set were 100 and 44%, respectively, at a score of 0 and 97 and 81% at a score of 1. Likelihood ratios (LRs) increased from 0 at a score of 0 to 40 at a score of > or = 4. CONCLUSIONS: Among children with CSF pleocytosis, a prediction score based on common tests of CSF and peripheral blood and intended for children with unavailable, negative, or delayed CSF Gram stain results has value for diagnosing bacterial meningitis.

Performance of rapid streptococcal antigen testing varies by personnel.

Rapid carbohydrate antigen tests are frequently used to diagnose group A streptococcal (GAS) pharyngitis. Despite evidence of modest sensitivity in medical settings, rapid antigen tests are available to the public for self-testing. We sought to determine if the personnel performing a rapid streptococcal antigen test influence the test's performance characteristics. Throat swabs of pediatric patients performed for GAS pharyngitis in a tertiary-care children's hospital network were included during two study periods in 2004 and 2005. The performance characteristics of a rapid carbohydrate antigen test were evaluated in three clinical settings against a nucleic acid probe test method according to the personnel performing the test (laboratory technologist versus nonlaboratory personnel). Between the study periods, nonlaboratory personnel from one site underwent retraining. Subsequently, the performance characteristics of the rapid antigen test were reassessed. The sensitivity of the rapid antigen test varied widely among the different testing sites (56 to 90%). Notably, test sensitivity was consistently greater when the test was performed by laboratory technologists than when it was performed by nonlaboratory personnel (P < 0.0001). Although the rapid antigen test sensitivity significantly improved after nonlaboratory personnel at one testing site were retrained (sensitivity before versus after retraining; P < 0.0001), the sensitivity remained greater in the laboratory technologist cohort (P < 0.0001). These data confirm the important relationship of the operator performing a rapid streptococcal antigen test with the test's accuracy, even in a clinical setting, where operator training is mandated. Therefore, its use outside the medical setting by lay persons cannot be recommended without culture backup.

Diagnosis of streptococcal pharyngitis by detection of Streptococcus pyogenes in posterior pharyngeal versus oral cavity specimens.

Carbohydrate antigen detection, nucleic acid probe detection, and bacterial culture are commonly used to confirm group A streptococcus (GAS) pharyngitis. Compared to standard throat swab specimens, the sensitivities of these tests with mouth specimens are poor. When testing for GAS pharyngitis, the throat remains the optimum site for sampling.

Evaluation of the Quidel QuickVue test for detection of influenza A and B viruses in the pediatric emergency medicine setting by use of three specimen collection methods.

The Quidel QuickVue influenza test was compared to viral culture and reverse transcriptase PCR by the use of three different respiratory specimen types. Of 122 pediatric subjects enrolled, 59 had influenza virus infections: 44 were infected with influenza A virus and 15 were infected with influenza B virus. The sensitivity of the QuickVue test was 85% with nasopharyngeal swabs, 78% with nasal swabs, and 69% with nasopharyngeal washes. Specificities were equivalent (97% to 98%) for all three collection methods.

Multicentre surveillance of the prevalence and molecular epidemiology of macrolide resistance among pharyngeal isolates of group A streptococci in the USA.

OBJECTIVES: Rates of macrolide resistance in group A streptococci (GAS) were reported to be low in the US in the 1990s. However, we documented an unexpectedly high rate of macrolide resistance among GAS in Pittsburgh, PA, in 2001 and 2002. In an effort to define the current prevalence of macrolide-resistant GAS in the US, a multicentre surveillance project was initiated. METHODS: Between October 2002 and May 2003, 50 pharyngeal GAS isolates per month were requested from each of the nine participating sites representing a wide geographical distribution. Standard susceptibility testing was performed and the macrolide resistance phenotype was assessed using double-disc diffusion testing. Monthly and annual rates of macrolide resistance were calculated for each site. An adjusted overall rate of macrolide resistance was determined to account for differences in the numbers of GAS isolates sent from each centre. RESULTS: Overall, 171 of the 2797 collected isolates of GAS (6.1%) were resistant to erythromycin. The adjusted overall resistance rate was 5.2%. Rates of macrolide resistance varied by site (range 3.0-8.7%) and also by month (<2% to >10%). The M phenotype of macrolide resistance accounted for >60% of all macrolide-resistant isolates recovered in this study. CONCLUSIONS: These data suggest an increasing prevalence and broad geographical distribution of macrolide-resistant GAS in the US, indicating the need for ongoing local and national longitudinal surveillance to define the extent of this problem.

In vitro activity of telithromycin against macrolide-susceptible and macrolide-resistant pharyngeal isolates of group A streptococci in the United States.

In vitro susceptibility testing of 2,797 group A streptococcus (GAS) isolates demonstrated that telithromycin was fully active against all macrolide-susceptible strains and among 80 of 115 macrolide-resistant GAS expressing the M phenotype. Telithromycin resistance was identified in 2 of 45 strains expressing the inducible macrolide-lincosamide-streptogramin B phenotype and four of nine isolates expressing the constitutive macrolide-lincosamide-streptogramin B resistance phenotype.