Development and performance of a multiplex PCR assay for the detection of bacteria in sterile body fluids.

Aim: To assess the performance characteristics of a lab-developed multiplex PCR assay for the detection of common bacterial pathogens associated with infections in pediatric patients from normally sterile sites, such as cerebrospinal fluid, synovial and pleural fluids. Materials & methods: A total of 272 specimens were tested by PCR and traditional culture methods to assess the presence of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Kingella kingae. Results: Compared with culture, the overall positive and negative percentage agreement of the PCR were 95.9% and 74.1%, respectively. Conclusion: This sterile body fluid PCR affords a rapid and sensitive alternative for bacterial detection, allowing for more timely pathogen-directed antimicrobial therapy.

Multiplex PCR Pathogen Detection in Acute Gastroenteritis Among Hospitalized US Children Compared With Healthy Controls During 2011-2016 in the Post-Rotavirus Vaccine Era.

BACKGROUND: Despite vaccine-induced decreases in US rotavirus (RV) disease, acute gastroenteritis (AGE) remains relatively common. We evaluated AGE pathogen distribution in hospitalized US children in the post-RV vaccine era. METHODS: From December 2011 to June 2016, the New Vaccine Surveillance Network (NVSN) conducted prospective, active, population-based surveillance in hospitalized children with AGE. We tested stools from 2 NVSN sites (Kansas City, Houston) with Luminex x-TAG Gastrointestinal Pathogen Panels (Luminex GPP) and analyzed selected signs and symptoms. RESULTS: For 660 pediatric AGE inpatients and 624 age-matched healthy controls (HCs), overall organism detection was 51.2% and 20.6%, respectively (P < .001). Among AGE subjects, GPP polymerase chain reaction detected >1 virus in 39% and >1 bacterium in 14% of specimens. Detection frequencies for AGE subjects vs HCs were norovirus (NoV) 18.5% vs 6.6%, RV 16.1% vs 9.8%, adenovirus 7.7% vs 1.4%, Shigella 4.8% vs 1.0%, Salmonella 3.1% vs 0.1%, and Clostridioides difficile in ≥2-year-olds 4.4% vs 2.4%. More co-detections occurred among AGE patients (37/660, 5.6%) than HCs (14/624, 2.2%; P = .0024). Per logistic regression analysis, ill contacts increased risk for NoV, RV, and Shigella (P < .001). More vomiting episodes occurred with NoV and RV, and more diarrheal episodes with Shigella and Salmonella. Modified Vesikari scores were highest for Shigella and lowest for C. difficile. CONCLUSIONS: NoV detection was most frequent; however, RV remained important in hospitalized AGE in the post-RV vaccine era. Continued active surveillance is important to document ongoing vaccine effects, pathogen emergence, and baseline disease burden for new vaccines.

Universal microbial diagnostics using random DNA probes.

Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD's unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics.

Performance of the Verigene® enteric pathogens test, Biofire FilmArray™ gastrointestinal panel and Luminex xTAG® gastrointestinal pathogen panel for detection of common enteric pathogens.

BACKGROUND: Multiplex syndromic panels have the capability of identifying causes of diarrheal illness. This study evaluated the performance characteristics of three multiplex molecular assays for the detection of common stool pathogens. METHODS: A total of 152 stool specimens were tested using three platforms: Verigene Enteric Pathogens Test (Verigene), Biofire FilmArray Gastrointestinal Panel (Biofire) and Luminex xTAG® Gastrointestinal Pathogen Panel (Luminex). Assays were assessed only for the targets common among all three; namely, Campylobacter, Salmonella, Shigella, Shiga toxin-producing E. coli (STEC), norovirus, and rotavirus. RESULTS: The sensitivities (%) and specificities (%) of the assays were: Campylobacter, Biofire (100,100), Verigene (83.3,99.3), Luminex (91.7,100); Salmonella, Biofire (95.8,100), Verigene (83.3,100), Luminex (79.2,100); Shigella, Biofire (100,100), Verigene (95.4,99.1), Luminex (100,100); STEC, Biofire (100,100), Verigene (91.7,100), Luminex (91.7,100); norovirus, Biofire (94.7,99.3), Verigene (89.0,100), Luminex (89.5,100); and rotavirus, Biofire (100, 98.6), Verigene (71.4,100), Luminex (100,100). CONCLUSIONS: All multiplex panels detected the majority of gastrointestinal pathogens when compared to conventional methods.

The human microbiome and its potential importance to pediatrics.

The human body is home to more than 1 trillion microbes, with the gastrointestinal tract alone harboring a diverse array of commensal microbes that are believed to contribute to host nutrition, developmental regulation of intestinal angiogenesis, protection from pathogens, and development of the immune response. Recent advances in genome sequencing technologies and metagenomic analysis are providing a broader understanding of these resident microbes and highlighting differences between healthy and disease states. The aim of this review is to provide a detailed summary of current pediatric microbiome studies in the literature, in addition to highlighting recent findings and advancements in studies of the adult microbiome. This review also seeks to elucidate the development of, and factors that could lead to changes in, the composition and function of the human microbiome.

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

Characterization of a unique ADP-ribosyltransferase of Mycoplasma penetrans.

Mycoplasma penetrans is a urogenital tract pathogen implicated in the deterioration of the immune system in human immunodeficiency virus-infected AIDS patients. Here, we describe a 78-kDa protein from M. penetrans, designated MYPE9110, that exhibits sequence similarity to known ADP-ribosyltransferases (ADPRTs) such as Bordetella pertussis pertussis toxin and Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin. MYPE9110 possesses key amino acid residues found in all ADPRTs that are essential for ADPRT activity. Several mammalian cell proteins are ADP-ribosylated by MYPE9110, and the full-length recombinant protein exhibits a strong auto-ADP-ribosylating activity. In the absence of target proteins, MYPE9110 demonstrates a NAD-glycohydrolase activity by hydrolyzing NAD. Furthermore, this toxin elicits cytopathology in HeLa cells by inducing cytoplasmic vacuolization in the presence of ammonium chloride. The deletion of the C-terminal region of MYPE9110 significantly diminishes its binding to host cells while still exhibiting an ADPRT activity, suggesting that MYPE9110 is a member of the family of A-B ADPRT toxins.