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Background: Ixodes scapularis ticks can carry Borrelia species as well as other pathogens that cause human disease. The frequency of tick-borne infections and coinfections in children with suspected Lyme disease is unknown, creating clinical uncertainty about the optimal approach to diagnosis. Methods: We enrolled children aged 1-21 years presenting to 1 of 8 Pedi Lyme Net emergency departments for evaluation of Lyme disease. We selected cases with serologically or clinically diagnosed Lyme disease (erythema migrans or early neurologic disease) matched by symptoms, age, gender, and center to control subjects without Lyme disease. We tested whole blood samples collected at the time of diagnosis using a multiplex high-definition polymerase chain reaction (HDPCR) panel to identify 9 bacterial or protozoan pathogens associated with human disease. We compared the frequency of tick-borne coinfections in children with Lyme disease to matched controls. Results: Of the 612 selected samples, 594 (97.1%) had an interpretable multiplex HDPCR result. We identified the following non-Borrelia tick-borne infections: Anaplasma phagocytophilum (2), Ehrlichia chaffeensis (1), and Babesia microti (12). Children with Lyme disease were more likely to have another tick-borne pathogen identified than matched controls (15/297 [5.1%] Lyme cases vs 0/297 [0%]; difference, 5.1% [95% confidence interval, 2.7%-8.2%]). Conclusions: Although a substantial minority of children with Lyme disease had another tick-borne pathogen identified, either first-line Lyme disease antibiotics provided adequate treatment or the coinfection was subclinical and did not require specific treatment. Further studies are needed to establish the optimal approach to testing for tick-borne coinfections in children.
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Early-onset neonatal sepsis due to Streptococcus agalactiae (group B Streptococcus [GBS]) infection is one of the leading causes of newborn mortality and morbidity. The latest guidelines published in 2019 recommended universal screening of GBS colonization among all pregnant women and intrapartum antibiotic prophylaxis for positive GBS. The updated procedures allow rapid molecular-based GBS screening using nutrient broth-enriched rectovaginal samples. Commercially available molecular assays for GBS diagnosis target mainly the cfb gene, which encodes a hemolysin protein responsible for producing the Christie-Atkins-Munch-Petersen (CAMP) factor. cfb is considered a conserved gene in essentially all GBS isolates. However, false-negative GBS results on Cepheid Xpert GBS and GBS LB tests due to deletions in or near the region that encodes cfb were reported recently. Therefore, the new Xpert GBS LB XC test was developed. This study is a multicenter evaluation of the new test for GBS identification from nutrient broth-enriched rectal/vaginal samples from antepartum women. A total of 621 samples were prospectively enrolled. The samples were tested with the Xpert GBS LB XC test, the composite comparator method, which included the Hologic Panther Fusion GBS test combined with bacterial culture, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and bacterial culture alone, followed by MALDI-TOF MS identification. The respective sensitivity and specificity of the Xpert GBS LB XC test were 99.3% and 98.7% compared to the composite comparator method and 99.1% and 91.8% compared to bacterial culture alone with MALDI-TOF MS identification. Overall, the Xpert GBS LB XC test performed comparatively to the composite comparator method and is equivalent to traditional bacterial culture followed by MALDI-TOF MS.
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Complicaciones Infecciosas del Embarazo , Infecciones Estreptocócicas , Recién Nacido , Embarazo , Femenino , Humanos , Complicaciones Infecciosas del Embarazo/diagnóstico , Complicaciones Infecciosas del Embarazo/microbiología , Vagina/microbiología , Streptococcus agalactiae/genética , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/microbiología , Sensibilidad y EspecificidadRESUMEN
Within healthcare settings, physicians use antibiograms, which offer information on local susceptibility rates, as an aid in selecting empirical antibiotic therapy and avoiding the prescription of potentially ineffective drugs. While antibiograms display susceptibility and resistance data at hospital, city, or region-specific levels and ultimately enable the initiation of antibiogram-based empirical antibiotic treatment, AST reports at the individual patient level and guides treatments away from broad-spectrum antibiotics towards narrower-spectrum antibiotics or the removal of antibiotics entirely. Despite these advantages, AST traditionally requires a 48- to 72-h turn-around; this window of time can be critical for some antimicrobial therapeutic interventions. Herein, we present a direct-from-specimen AST to reduce the time between patient sampling and receipt of lab AST results. The biggest challenge of performing AST directly from unprocessed clinical specimens with an unknown microbial load is aligning the categorical susceptibility report with CLSI reference methods, which start from a fixed inoculum of 0.5 McFarland units prepared using colonies from a sub-culture. In this pilot clinical feasibility study using de-identified remnant specimens collected from MCW, we observed the high and low ends of microbial loads, demonstrating a final categorical agreement of 87.5% for ampicillin, 100% for ciprofloxacin, and 100% for sulfamethoxazole-trimethoprim.
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Antibacterianos , Ciprofloxacina , Humanos , Estudios de Factibilidad , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ciprofloxacina/farmacología , Ciprofloxacina/uso terapéutico , AmpicilinaRESUMEN
OBJECTIVES: We conducted an analytic and clinical comparison of a novel high-definition polymerase chain reaction PCR (HDPCR) assay to traditional real-time PCR (RT-PCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper respiratory specimens. METHODS: Analytic performance of RT-PCR, HDPCR, and extraction-free HDPCR was established through replicate testing of a serially diluted clinical specimen containing SARS-CoV-2. A clinical comparison of all 3 assays was conducted using 351 prospectively collected upper respiratory swab specimens obtained from symptomatic and asymptomatic individuals collected in various transport media. RESULTS: RT-PCR and HDPCR assays using extracted nucleic acid demonstrated similar analytic limits of detection (LoD) and clinical performance, with 100% positive and negative agreement. Extraction-free HDPCR demonstrated a 1.5 to 2.0 log10 increase in LoD based on cycle threshold values. However, clinical performance of extraction-free HDPCR remained high, demonstrating 97.8% positive and 99.6% negative agreement with RT-PCR. An overall increase in "invalid" and "presumptive" results was observed when using the extraction-free method, but this was highly variable based on transport medium used. CONCLUSIONS: HDPCR performs similar to RT-PCR for the detection of SARS-CoV-2. The use of an extraction-free HDPCR protocol maintained high clinical performance despite reduced analytic LoD, with the benefit of reduced hands-on time and cost of reagents associated with nucleic acid extraction.
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COVID-19/genética , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genética , COVID-19/diagnóstico , Prueba de COVID-19/métodos , Humanos , Límite de Detección , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/patogenicidad , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
NG-Test Carba 5 is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of five common carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM) directly from bacterial colonies. The assay is simple to perform and has recently received U.S. Food and Drug Administration clearance. A method comparison study was performed at geographically diverse medical centers (n = 3) in the United States, where 309 Enterobacterales and Pseudomonas aeruginosa isolates were evaluated by NG-Test Carba 5 (NG Biotech, Guipry, France), the Xpert Carba-R assay (Cepheid, Inc., Sunnyvale, CA), the modified carbapenem inactivation method (mCIM), the EDTA-modified carbapenem inactivation method, and disk diffusion with carbapenems. Colonies from tryptic soy agar with 5% sheep blood (blood agar) and MacConkey agar were tested, and the results were compared to those obtained by a composite reference method. Additionally, a fourth medical center performed a medium comparison study by evaluating the performance characteristics of NG-Test Carba 5 from blood, MacConkey, and Mueller-Hinton agars with 110 isolates of Enterobacterales and P. aeruginosa These results were compared to the expected genotypic and mCIM results. For the multicenter method comparison study, the overall positive percent agreement (PPA) and the overall negative percent agreement (NPA) of NG-Test Carba 5 with the composite reference method were 100% for both blood and MacConkey agars. The medium comparison study at the fourth site showed that the PPA ranged from 98.9% to 100% and that the NPA ranged from 95.2% to 100% for blood, MacConkey, and Mueller-Hinton agars. NG-Test Carba 5 accurately detected and differentiated five common carbapenemase families from Enterobacterales and P. aeruginosa colonies on commonly used agar media. The results of this test will support a streamlined laboratory work flow and will expedite therapeutic and infection control decisions.
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Proteínas Bacterianas , beta-Lactamasas , Animales , Proteínas Bacterianas/genética , Francia , Sensibilidad y Especificidad , Ovinos , beta-Lactamasas/genéticaRESUMEN
The prevalence of tick-borne infections has been steadily increasing in both number and geographic distribution in the United States and abroad. This increase, in conjunction with the continued recognition of novel pathogens transmitted by ticks, has made accurate diagnosis of these infections challenging. Mainstay serologic tests are insensitive during the acute phase of infection and are often cross-reactive with similar pathogenic and nonpathogenic organisms. Further, they are unable to reliably differentiate active versus past infection which can lead to misdiagnosis and incorrect understanding of the epidemiology and incidence of specific tick-borne pathogens. We evaluated a novel multiplexed high-definition PCR (HDPCR) Tickborne Panel (TBP) assay (ChromaCode, Carlsbad, CA) for the detection of nine tick-borne pathogens or groups associated with human illness. The HDPCR technology enables multiplex identification of multiple targets in a single fluorometric channel based on fluorescent signal modulation using a limiting probe design. A collection of 530 whole-blood specimens collected from patients being evaluated for tick-borne infections, in addition to a panel of 93 simulated specimens, were used to challenge the HDPCR TBP. The results were compared to a clinically validated traditional multiplexed PCR test with additional sequence analysis and clinical history collected to aid in resolving discrepancies. Among clinical specimens the TBP demonstrated 100% sensitivity for the identification of Anaplasma phagocytophilum, Borrelia miyamotoi, Borrelia mayonii, and Rickettsia rickettsii The sensitivity for identification of B. burgdorferi was 44.4% compared to a composite gold standard. Among simulated specimens containing single or multiple targets present at 103 to 105 copies/PCR, the sensitivity of TBP was 100% for all targets, with a combined specificity of 99.5%. Of note, an increased rate of false-positive results was observed among simulated specimens that contained multiple targets. Based on these data, we find the HDPCR TBP to be a useful adjunct for the diagnosis of tick-borne infections in patients with suspected tick-borne illness.