Jeanne A. Jordan

Vaginal swabs are the specimens of choice when screening for chlamydia trachomatis and Neisseria gonorrhoeae : Results from a multicenter evaluation of the APTIMA assays for both infections

Background: Vaginal swabs were recently U.S. Food and Drug Administration-cleared for detecting Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) using Gen-Probe Incorporated’s APTIMA COMBO2 Assay (AC2). We assessed the APTIMA CT Assay (ACT) for CT, APTIMA GC Assay (AGC) for GC, and AC2 for both organisms using patient- and clinician-collected vaginal swabs. Method: Women attending family planning, obstetrics and gynecology, or sexually transmitted disease (STD) clinics had first-catch urines (FCUs), patient-collected vaginal swabs, clinician-collected vaginal swabs, and endocervical swabs tested by ACT, AGC, and AC2. A second endocervical swab and FCU were tested using BD ProbeTec (Becton Dickinson) for CT and GC. We calculated sensitivity and specificity using vaginal swabs to detect CT and GC. Results: Of 1464 subjects enrolled, 180 had CT and 78 GC. ACT sensitivities and specificities for patient-collected vaginal swabs were 98.3% and 96.5%, respectively; for clinician-collected vaginal swabs, 97.2% and 95.2%, respectively. AGC sensitivities and specificities for patient-collected vaginal swabs were 96.1% and 99.3%, respectively; for clinician-collected vaginal swabs, 96.2% and 99.3%, respectively. AC2 results were similar. If an FCU tested positive for CT or GC, >94% of matching vaginal swabs were positive. Positive endocervical swabs showed slightly less concordance (>90% and >88%, respectively). More infected patients were identified using vaginal swabs than FCUs. With AC2, 171 CT-infected patients were identified using FCUs and 196 using patient-collected vaginal swabs. This difference was more pronounced for CT than for GC. Conclusions: Vaginal swab specimens allowed sensitive and specific detection of CT and GC in the APTIMA assays. Vaginal swabs identified as many infected patients as endocervical swabs and more than FCUs, and may well be the specimen of choice for screening.

Hemodynamic disturbances in premature infants born after chorioamnionitis: Association with cord blood cytokine concentrations

Chorioamnionitis and elevated cord blood inflammatory cytokine concentrations are associated with detectable disturbances of systemic and cerebral hemodynamics in premature newborns. Fifty-five infants (25–31 wk gestation) were enrolled. Chorioamnionitis was defined by placental histology. IL-6, IL-1β, and tumor necrosis factor-α were quantified by ELISA. Blood pressure, heart rate, cardiac output, stroke volume, fractional shortening, and middle cerebral artery blood flow velocities were measured at 3 ± 1 h after birth. Chorioamnionitis was evident in 22 placentas and was associated with increased IL-6 (p < 0.001), IL-1β (p = 0.035), and heart rate (p = 0.027); and with decreased mean and diastolic blood pressure (p = 0.026 and p = 0.019, respectively). IL-6 concentration correlated inversely with systolic, mean, and diastolic blood pressures. Right ventricular cardiac output was elevated (p = 0.028) in infants with fetal vessel inflammation. Maternal temperature ≥38.0°C and newborn immature-to-total white blood cell ratio ≥0.4 were associated with significant decreases in left ventricular fractional shortening (p = 0.001 and p = 0.005, respectively). Neither chorioamnionitis nor elevated cytokine concentrations were associated with changes in middle cerebral artery Doppler blood flow velocities. Chorioamnionitis and elevated cord blood IL-6 concentrations are associated with decreased blood pressure in premature newborns. Inflammation of the fetal vessels and nonspecific indicators of infection are associated with disturbances in cardiac function. Infants with chorioamnionitis and elevated cytokine concentrations do not manifest changes in cerebral Doppler indices within the first few postnatal hours. We speculate that cytokine-associated systemic hemodynamic disturbances in premature infants born after chorioamnionitis predispose such infants to perinatal brain injury.

Women find it easy and prefer to collect their own vaginal swabs to diagnose Chlamydia trachomatis or Neisseria gonorrhoeae infections.

Background: Self-collected specimens can be used to screen asymptomatic women for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). We surveyed women’s opinions on ease and preferences as to sampling after collecting their own vaginal swab and urine and a physician collection of vaginal swab and cervical swab. Methods: In 7 North American cities, a questionnaire was used for women after they participated in a clinical trial of nucleic acid amplification testing of various specimens. A total of 1090 women consenting to gynecologic sampling for CT and GC (82% of those sampled) volunteered to complete the survey. We analyzed the data for ease of self-collection and preferences for a vaginal swab, urine, or cervical swab. Results: The average age was 26.6 years; 59.6% were black, 25.5% white, 11% Hispanic, 1.9% Asian, and 2% unknown. Thirty-five percent had more than one sex partner in the past 6 months, 84.9% had been previously tested for a sexually transmitted infection (STI), and 49.2% had experienced an STI. A total of 90.4% found it very easy to self-collect a vaginal swab. This was not influenced by age, education, or study site. Seventy-six percent preferred a vaginal swab over a pelvic examination, 60% over a urine collection, and 94% indicated that they would be tested more often if a vaginal swab was available. Conclusion: Self-collected vaginal swabs were easy to collect and patients preferred them over urine and cervical swabs.

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

Speed is of the essence when evaluating an infant with symptoms consistent with sepsis. Because of the high morbidity and mortality associated with neonatal sepsis, infants receive multiple, broad-spectrum antibiotics before receiving finalized blood culture results. Incorporating an additional, reliable, yet rapid assay to detect bacteria directly from blood would facilitate timely diagnosis and appropriate care. To this end, we designed a real-time polymerase chain reaction (PCR) assay targeting the highly conserved 380 bases of 16S rDNA. DNA was extracted from whole-blood samples using a Qiagen column. The limit of detection for the TaqMan-based assay, using a Smartcycler instrument, was 40, 50, or 2000 colony-forming units per milliliter of blood (CFU/ml) of Escherichia coli, group B Streptococcus, and Listeria monocytogenes, respectively, when white blood cell counts were below 39,000/microl. Implementing this approach requires less than 4 hours for both sample preparation and real-time PCR compared with 1 to 2 days to detect growth in culture or 5 days to finalize no-growth culture results. There was an overall agreement between the results of culture and real-time PCR of 94.1% (80 of 85) in this study. These results suggest that molecular techniques can augment culture-based methods for diagnosing neonatal sepsis, especially in infants whose mothers have received intrapartum antibiotic prophylaxis.

Use of pyrosequencing of 16S rRNA fragments to differentiate between bacteria responsible for neonatal sepsis.

Infants admitted to neonatal intensive care units for suspicion of bacterial sepsis receive at least two broad-spectrum antibiotics for a minimum of 48 to 72 hours to cover both gram-positive and gram-negative organisms while awaiting blood culture results. On average, bacterial growth becomes detectable within 12 to 24 hours, with an additional 24 to 48 hours required for identification. We have previously described using a 16S rRNA PCR assay for screening neonatal blood for bacterial DNA. Combining PCR with DNA sequencing could prove a faster means of detecting bacteria than culture-based identification. If successful, antibiotic therapy could be appropriately tailored sooner, thus sparing infants the administration of unnecessary antibiotics. Our goal was to assess the potential of pyrosequencing to differentiate between bacteria commonly associated with neonatal sepsis. To begin, full-length sequencing of the 380-bp 16S rRNA amplicons from representative bacteria was conducted (ABI 3100) and several databases queried. These included Staphylococcus sp., Streptococcus sp., Listeria sp., and numerous gram-negative rods. The sequences from clinical isolates were identical to those present in the published databases for the same bacteria. As a result, an informative 15 bases within the 380-bp amplicon was targeted for pyrosequencing following enrichment culture and PCR amplification. A total of 643 bacterial isolates commonly associated with neonatal sepsis, and 15 PCR-positive, culture-positive neonatal whole blood samples were analyzed by pyrosequencing. Results of DNA sequencing and culture identification were compared. In summary, we were successful at using PCR and pyrosequencing together to accurately differentiate between highly diverse bacterial groups.

Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles

ABSTRACT Growth in liquid media is the gold standard for detecting microorganisms associated with bloodstream infections. The Gram stain provides the first clue as to the etiology of infection, with phenotypic identification completed 1 or 2 days later. Providing more detailed information than the Gram stain can impart, and in less time than subculturing, would allow the use of more directed empirical therapy and, thus, reduce the patients exposure to unnecessary or ineffective antibiotics sooner. The study had two objectives, as follows: (i) to identify new targets to improve our ability to differentiate among certain enteric gram-negative rods or among certain Streptococcus species and (ii) to determine whether real-time PCR and pyrosequencing could as accurately identify organisms directly from positive blood culture bottles as culture-based methods. Two hundred and fifty-five consecutive positive blood culture bottles were included. The results showed a high level of agreement between the two approaches; of the 270 bacteria isolated from the 255 blood culture bottles, results for pyrosequencing and culture-based identifications were concordant for 264/270 (97.8%) bacteria with three failed sequences, and three sequences without match. Additionally, compared to the universal 16S rRNA gene target, the new 23S rRNA gene targets greatly improved our ability to differentiate among certain enteric gram-negative rods or among certain Streptococcus species. In conclusion, combining real-time PCR and pyrosequencing provided valuable information beyond that derived from the initial Gram stain and in less time than phenotypic culture-based identification. This strategy, if implemented, could result in a more directed empirical therapy in patients and would promote responsible antibiotic stewardship.

Ability of New APTIMA CT and APTIMA GC Assays To Detect Chlamydia trachomatis and Neisseria gonorrhoeae in Male Urine and Urethral Swabs

ABSTRACT A clinical evaluation was conducted in six North American centers to determine the ability of APTIMA CT (ACT) and APTIMA GC (AGC) nucleic acid amplification assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections in 1,322 men by testing their urethral swabs and first-catch urine (FCU). The results obtained with ACT and AGC assays were compared to an infected patient status determined by testing the specimens with the APTIMA Combo 2 and the BD ProbeTec energy transfer multiplex assays. Symptoms did not influence the values. Positive and negative agreements of the ACT and AGC assays for individual specimens were high, with each comparator assay ranging between 94.3 and 100% for positives and 93.9 and 99.4% for negatives. The ACT and AGC assays performed on noninvasive specimens such as FCU effectively identified C. trachomatis or N. gonorrhoeae infections in symptomatic and asymptomatic men and should be suitable for screening male populations.

Confirming Positive Results of Nucleic Acid Amplification Tests (NAATs) for Chlamydia trachomatis: All NAATs Are Not Created Equal

ABSTRACT The Centers for Disease Control and Prevention recommended confirming positive screening tests for Chlamydia trachomatis when positive predictive values are <90%. It is accepted that less sensitive tests (i.e., culture and immunoassays) should not be used to confirm the results of more sensitive nucleic acid amplification tests (NAATs). We show that the same principle applies when NAATs are used for confirmation.

A call to action for antimicrobial stewardship in the emergency department: approaches and strategies.

Antimicrobial resistance is a mounting public health concern. Emergency departments (EDs) represent a particularly important setting for addressing inappropriate antimicrobial prescribing practices, given the frequent use of antibiotics in this setting that sits at the interface of the community and the hospital. This article outlines the importance of antimicrobial stewardship in the ED setting and provides practical recommendations drawn from existing evidence for the application of various strategies and tools that could be implemented in the ED including advancement of clinical guidelines, clinical decision support systems, rapid diagnostics, and expansion of ED pharmacist programs.

Rapid group B streptococci screening using a real-time polymerase chain reaction assay

OBJECTIVE: To estimate the clinical performance characteristics of a real-time polymerase chain reaction (PCR) assay using vaginal/rectal swabs from antepartum (35–37 weeks of gestation) and intrapartum women. METHODS: The assay evaluated is a qualitative, automated, real-time PCR test for the detection of group B streptococci, with results available in approximately 75 minutes. Enrollment in this multicenter clinical study occurred between October 2005 and January 2006. Vaginal/rectal swabs were analyzed by nursing personnel (intrapartum tests) or by laboratory technologists (all others). Polymerase chain reaction assay results were compared with culture using standard methods, including selective broth medium, and to a predicate nucleic acid amplification test. RESULTS: Of 1,028 enrolled women, 234 were deemed ineligible, and 10 had unresolved test results. Of the 784 remaining women, valid PCR assay results were obtained on the first test attempt for 93.0%. Performance characteristics relative to culture were sensitivity 91.1%, specificity 96.0%, positive predictive value 87.8%, negative predictive value 97.1%, and accuracy 94.8%. These results exceeded those obtained using the predicate nucleic acid amplification test. CONCLUSION: Performance characteristics of the PCR assay exceed the threshold recommended by the Centers for Disease Control and Prevention when compared with culture. The test is sufficiently robust to be performed for intrapartum patients in a point-of-care setting by medical professionals. LEVEL OF EVIDENCE: II