The evaluation of children presenting with urinary tract infection (UTI) has long entailed sonography and cystography to identify all urological abnormalities that might contribute to morbidity. The identification of vesicoureteral reflux (VUR) has been of primary concern since retrospective studies from the 1930s to 1960s established a strong association between VUR, recurrent UTI, and renal cortical scarring. It has been proposed that all VUR carries a risk for renal scarring and, therefore, all VUR should be identified and treated. We will not discuss the controversies surrounding VUR treatment in this review focusing instead on a new paradigm for the evaluation of the child with UTI that is predicated on identifying those at risk for scarring who are most deserving of further evaluation by cystography. Concern for the influence of vesicoureteral reflux (VUR) on renal cortical scarring began with the recognitionthat VUR transmits bacteria to the upper urinary tract and the observation thatAPN, VUR, and renal scarring frequently coexisted in the same patient. AlthoughAPN was considered to be an ascending infection, it was mistakenly considered to alwaysresult from VUR , 2. WithSonography is appealing as a method ofevaluation following UTI; however, its ability to detect upper urinary tractabnormalities is limited. Although RBUS is noninvasive and sufficientlysensitive to evaluate collecting system dilatation, controlled trials havefound it to be less sensitive than contrast cystography and99m-Tc-dimercaptosuccinic acid (DMSA) renal scintigraphy in detecting VUR orparenchymal lesions. As much as 60% ofreflux and 50% of renal scan abnormalities noted on DMSA are routinely missedby sonography –6. GivenExperimental studies in the refluxingpiglet model of ascending acute pyelonephritis were conducted using stricthistopathologic criteria as the standard of reference to evaluate the truesensitivity and specificity of renal cortical scintigraphy , 10. TheThe DMSA scan was determined to behighly sensitive and reliable for the detection and localization ofexperimental acute pyelonephritis, with a sensitivity of 87 to 89 percent andspecificity of 100 percent for both. When individual pyelonephritic lesionswere analyzed, DMSA scan findings correlated with histopathological changes with an overallagreement rate of 89 to 94 percent. Those lesions not detected were microscopicfoci of inflammation not evident on gross examination and not associated withsignificant parenchymal damage. DMSA nuclearrenography has been shown to be the most accurate (96%) for the detection ofAPN as compared with gadolinium-enhanced MRI (91%), contrast CT scan (90%), andpower Doppler sonography (69%) .The clinical diagnosis of acutepyelonephritis traditionally has been made on the basis of the classic signsand symptoms of fever and flank pain or tenderness associated with pyuria andpositive urine culture. However, accurate diagnosis based solely on theseparameters is often incorrect, particularly in neonates and infants . DespiteThe critical role that infection playsin the pathogenesis of renal scarring associated with VUR was clarified byRansley and Risdon's experimental studies on the refluxing piglet model, bywhich—in the face ofVUR and normal voiding pressures—they showedthat renal scarring occurs only when UTI is present . While aSeveral investigators have nowevaluated the evolution of the acute inflammatory changes associated withpyelonephritis using serial DMSA renal scans –27. Acutattributable in part tothe increased risk of these kidneys for acute inflammatory damage at the timeof the initial infection [Despite these findings, the importanceof VUR (particularly grades III or higher) as a risk factor for renal scarringshould not be discounted. Clearly, patients with moderate and severe reflux aremuch more likely to develop acute pyelonephritic damage than children with mildor no reflux , 29. Furnfection , 28, 30.The DMSA renal scan could supplant thestandard evaluation scheme since it can better discriminate which child is atrisk for renal scarring irrespective of the presence of VUR. In this novel algorithm, a child who presentswith clinical signs and symptoms suggestive of pyelonephritis first undergoes aDMSA renal scan to detect renal inflammation. Once radiographic evidence ofrenal involvement is demonstrated, the child then undergoes cystography tosearch for VUR. This approach is supported by the following two studies.Hansson et al. retrospectively reviewed 303 children under 2years of age who presented with a first UTI (82% of whom had fever onpresentation) . VUR wasIn a follow-up study, Preda et al. prospectively evaluated290 children less than 1 year of age with UTI (79% of whom had fever onpresentation) with VCUG and DMSA renal scans . Fifty-oThis algorithm is further supported bythe observations that while clinically significant acute lesions may occur inthe presence or absence of VUR, significant renal scarring is most likely tooccur in those with gradesIII and IV VUR . Taken tThe “top-down” approach also serves toreduce the overall charges for the evaluation of children with UTIs. How greata reduction depends on what estimates are used for the prevalence of patientswith VUR among children with febrile UTI, and among those with positive DMSArenal scans as well as the timing of evaluation. DMSA scans performed at alonger interval following the acute episode are less likely to demonstratepyelonephritis, as many as half of those lesions having resolved. In In our effort to identify those children who aremost at risk, we might be tempted to apply certain demographic facts to furtherminimize studies. Patient age is recognized as being a risk factor for renalscarring; younger individuals are thought to be more susceptible. Gender mayalso play a role as younger males, particularly those who are not circumcised,tend to be at greater risk for UTI as well as high grade reflux. Reflux inblacks is also less common; however the renal effects are the same as in othergroups in those who have reflux . FinallyTherefore, a negative evaluation by anymeans, whether by the traditional approach or by the “top-down” approach, maynot be sufficient to exclude all children at risk. Therefore, those who presentwith recurrent febrile UTI deserve further attention. We, therefore, recommend that a prudentapproach includes DMSA renal scans for all children with febrile UTI, ideallyas close to the acute episode as possible. Those with positive scans shouldundergo cystography. If VUR is identified, the clinician should then formulatean appropriate treatment plan depending on various factors such as likelihoodof VUR resolution, likelihood of renal scarring, taking into considerationparental concerns regarding various treatments, and their outcomes. Childrenwith a negative DMSA scan require no further evaluation unless recurrentfebrile UTI occurs, in which case cystography should be performed. The use ofantimicrobial prophylaxis should be recommended based on known risks forrecurrent UTI and for renal scarring should UTI occur regardless of thepresence of absence of reflux.The “top-down” evaluation provides anassessment of risk for renal scarring that strongly correlates with thepresence of clinically relevant VUR. Inaddition to being a more cost-effective means of evaluating children withfebrile UTIs, it is also predicted to lessen medical care costs by reducing thenumber of children on antibiotic prophylaxis and of children undergoing surgeryfor reflux that is unlikely to result in renal scarring.


Vesicoureteral reflux (VUR) is the most common anomaly associated with duplex systems. In addition to an uncomplicated duplex system, reflux can also be secondary in the presence of an ectopic ureterocele with duplex systems. Controversy exists in regard to the initial and most definitive management of these anomalies when they coexist. This paper will highlight what is currently known about duplex systems and VUR, and will attempt to provide evidence supporting the various surgical approaches to an ectopic ureterocele and duplex system and the implications of concomitant VUR. Less than 1% of thegeneral population has a duplex kidney . VUR is There are certainfactors that contribute to reflux resolution in single-system (SS) ureters,including patient age, grade of reflux, postnatal presentation, and thepresence or absence of associated voiding dysfunction . The natData from the availableliterature suggests that the majority of patients with DS and low-grade VUR canbe initially managed with antibiotics and careful observation. Parents should be counseled that it may takelonger for the reflux to resolve and young females with high-grade VUR may beat increased risk for infections. Despite these findings, the absolute indication for surgery inindividuals with low-grade VUR is not different from those with SS and similar VUR,and surgical correction is successful in the majority of cases . In factDuplex systems are anuncommon diagnosis causing prenatal hydronephrosis; however, when confirmed,ureteroceles are one of the most common associated findings , 10. EctThe initial and subsequent management of ureteroceles has beencontroversial and depends on several factors, including presenting symptoms, ectopicversus orthotopic position, presence or absence of reflux, and function of theassociated upper-pole moiety . Asthe In the above proposed setting, the clear indication for endoscopicdecompression of an ectopic ureterocele is in a child who presents with sepsis orbladder outlet obstruction. However, in thesetting of sepsis, one must open the ureterocele completely, as puncturing maynot result in adequate drainage. Thisprocedure almost invariably results in prompt improvement in patient symptoms,but the parents should be counseled that their child will require definitive reconstructionat a later date, as reflux into the upper-pole moiety is the rule, not theexception. In contrast, endoscopicpuncture of an ectopic ureterocele in the nonemergent setting may also committhe patient to future reconstruction. Inone series describing endoscopic puncture for ectopic ureteroceles, Jayanthi etal. reported postoperative reflux into the upper-pole moiety in 50% of cases . OverallUpper-pole heminephrectomy can result in excellent decompression of theureterocele and should be the procedure of choice if there is no ipsilaterallower pole or contralateral reflux . RemovinIn conclusion, ectopic ureteroceles that reflux or are associated withreflux into other moieties are likely best served with ureterocele excision ormarsupialization, bladder floor reconstruction, and ureteral reimplant. Another option would be a ureteroureterostomywith a lower-pole extravesical reimplant. In those patients who present with sepsis or bladder outlet obstruction,endoscopic decompression is highly successful but will likely commit thepatient to further surgery. Upper-poleheminephrectomy is best applied to those patients with nonfunctioning upperpoles and no associated reflux. In thissetting, this approach is highly successful and has the advantage of avoidingbladder surgery, limiting risks to the lower pole, and eliminating thepotential unknown risks of preserving a dysplastic upper pole . ArguablReflux found inassociation with a duplex system may take longer to resolve than single-systemreflux. Parents should be counseledaccordingly. Surgery to correct VUR induplex systems is highly successful. Ectopicureteroceles can present an interesting and difficult surgical challenge andcan be ultimately managed with multiple surgical approaches following initialconservative therapy. Endoscopicdecompression seems best reserved for the septic patient or one who presentswith bladder outlet obstruction. Itprovides excellent relief of obstruction and can preserve upper-pole renalfunction. Ultimately, these patients arecurrently managed by either an upper- or lower-tract approach. The most important factor in deciding whichapproach to take is the presence or absence of VUR.


This case is about a 48-year-old woman known with a reduced butyrylcholinesterase activity, who developed prolonged neuromuscular blockade following the unintentional administration of succinylcholine. We took the opportunity to monitor the development of neuromuscular function during this period and blood samples were taken for molecular genetic analysis and for quantitative and qualitative analysis since not all causative mutations are functionally characterized. Reduced butyrylcholinesterase activity is discussed in many aspects. Clinical considerations are suggested concerning genetic counselling. Normally 90 percent of intravenously administered succinylcholine is hydrolysed in the plasma by butyrylcholinesterase (BCHe) before it can bind to the alpha subunits of the nicotinic cholinergic receptor at the neuromuscular junction. BCHe hydrolyses ester bonds in many agents; local anaesthetics-like cocaine, procaine, chloroprocaine, and tetracaine, and also heroine and mivacurium, are metabolised by BCHE. Decreased activity of BCHe becomes clinically relevant in patients carrying at least one causative mutation in the BCHE gene . The BCHFollowing administration of succinylcholine to a patient with a known abnormal variant of BCHe, the duration of relaxation of skeletal muscles is predictable. In this case history the genotype of the patient was not known yet. After informed consent obtained from the patient we describe this case history concerning prolonged neuromuscular blockade following succinylcholine administration to a patient with a reduced butyrylcholinesterase activity and the follow-up investigation.A 48-year-old woman  was scheduled for a laparoscopic cholecystectomy because of cholelithiasis. At the preoperative assessment she reported postoperative apnoea following a Caldwell-Luc procedure at the ENT-OR in 1981. A prolonged effect of succinylcholine had been confirmed by a dibucaine number of 10, written on a crumpled warning card. The patient herself was absolutely clear about the condition of her butyrylcholinesterase deficiency. Her medical history showed an appendectomy, hypertension, and a trigeminal neuralgia due to repetitive sinus infections. Current medication was amitriptyline, gabapentin, diclofenac, omeprazole, metoprolol, and acetaminophen combined with codeine. Induction of anaesthesia was carried out with fentanyl and propofol. To facilitate tracheal intubation 100 mg succinylcholine (0.9 mg/kg) had been administered unintentionally. Maintenance was performed with intermittent dosages of fentanyl, morphine and a continuous infusion of propofol. The surgical procedure lasted 90 minutes and was done uneventfully.Monitoring of neuromuscular function was started using acceleromyography (TOF-Watch SX), by performing intermittently a “train-of-four”. Determination of the supramaximal stimulus could not be performed, as deep neuromuscular blockade was already induced. Anaesthesia, using propofol continuously in combination with intermittent doses of morphine, and neuromuscular monitoring were continued until recovery of the TOF-ratio above 90%; at this value it was assumed that the patient could breathe without help. Recovery of neuromuscular function took more than seven hours. The first twitch was seen 87 minutes after the administration of succinylcholine. The course was uneventful although the patient was emotional and anxious postoperatively we were able to regain her trust after several talks.After informing this adverse event to the patient and her partner, she gave permission to take blood samples for quantitative and qualitative analysis and for molecular genetic investigation.The plasma concentration of cholinesterase was 2520 U/l (ref: 4800–12000 U/l) and the dibucaine number was 8. These results are indicative for a homozygous atypical variant. Because of the very long clinical duration of action we did not limit molecular genetic investigations to known BCHE variants but decided to sequence both strands of the complete BCHE coding sequence. This identified a homozygous A- as well as a homozygous K-variant. The A-variant  is most causative for the clinical feature and the K-variant  contributed to the markedly prolonged muscle relaxation and the dibucaine number of 8. The clinical significance of the K-variant as a single mutation has been described by Gätke et al. [Immediately after induction, the attending anaesthesiologist realized that he had made a big mistake most probably caused by overconcentration. Although all preparations were done, in word and writing, this case shows us that anesthesia is done by humans. Unfortunately man makes mistakes. The best thing you can do afterwards is to be honest to the patient and that you will try to avoid it in the future.Succinylcholine in a dosage of 1 mg/kg has a rapid onset (30–60 sec.) and a short duration of action (3–5 min.). It mimics the action of acetylcholine at the neuromuscular junction by depolarising the postjunctional membrane, provided that the depolarisation is sustained until succinylcholine is hydrolysed by BCHe. BCHe is synthesised by the liver, and among other plasma cholinesterases its physiological function is still not fully understood. In 1957, Werner Kalow identified plasma cholinesterase (pseudocholinesterase) as the cause of variable metabolism of drugs when he described persisting neuromuscular blockade (apnoea) after the administration of succinylcholine . By inveGenetic variants of butyrylcholinesterase result in differences in clinical response to succinylcholine. This often unexpected drug effect with potential morbidity as a consequence makes decreased BCHe activity a pharmacogenetic disorder . The actIn response to an adverse event in which an abnormal BCHe activity is suspected the first step is to perform biochemical investigations to confirm the phenotype . To specThe A- variant we detected  is different from the most frequent occurring A variant . Diagnosing the mutations in the BCHE gene in our patient, we could offer family members genetic counselling to screen for this mutation [In conclusion, using neuromuscular blocking agents, it is recommended to monitor the recovery of neuromuscular function. Persisting neuromuscular blockade following the administration of succinylcholine should be investigated using conventional biochemical testing and molecular genetic testing to distinguish between an inherited or acquired cause for the decreased activity of BCHe. Applying this knowledge into clinical practice will improve the safety of our patients and their relatives.


The aim of this study was to assess the responsiveness of the asthma control test (ACT) to detect changes at the initiation of therapy and its utilization in the initiation of asthma treatment.This study was designed as a randomized clinical trial conducted in a primary care setting. The subjects were asthma patients who had not received controller therapy for at least two months. The patients were randomized into two groups: The Saudi Initiative for Asthma (SINA) group and the Global Initiative for Asthma (GINA) group. Treatment in the SINA group was initiated at step1 when the ACT scores ≥ 20, step 2 when the score between16-19, and step 3 when the score < 16 began at step 3. The GINA group patients were started on step 2 when they had persistent asthma symptoms or step 3 when they had severely uncontrolled disease.p = 0.04)). The improvement in FEV1 was 5.8% in the SINA group compared to 3.4% in the GINA group (p = 0.46). The number of patients who achieved asthma control at the follow-up visit and required no treatment adjustment was 33 (73.3%) in the SINA group and 27 (60%) in the GINA group (p = 0.0125).Forty-five patients were analyzed in each group. The improvement in ACT score after treatment initiation was significantly higher when the SINA approach was used (2.9 in the SINA group compared to 1.7 in the GINA group (The ACT was responsive to change at the initiation of asthma treatment and was useful for the initiation of asthma treatment.ISRCTN31998214 The Global Initiative for Asthma (GINA) has evolved management from being based on a severity index to the concept of achieving asthma control -4. It haThe Saudi Initiative for Asthma management (SINA) was created by the Saudi Thoracic Society which was adopted and customized from the GINA, the NAEPP, and the available local literature ,8. The SThis study was a randomized clinical trial conducted in asthma patients who presented at primary care centers belongs King Abdulaziz Medical City, Riyadh, Saudi Arabia between 21 September and 12 November 2011. These primary care centers were operated independent of the main hospital and open to patients who has medical records at the center. The inclusion criteria included the following: an age above 12 years, a diagnosis of asthma, and literacy, as patients had to answer the ACT without assistance. Patients were excluded if they had used controller therapy for the two months prior to the initial presentation in order to ensure that our findings are contaminated by a prior controller treatment. Controller therapy was defined as the use of inhaled corticosteroids, leukotriene modifiers, and/or long-acting bronchodilators agents. This study was approved by the Institutional Review Board of the King Abdullah International Center for Medical Research (RC10-091). It was also registered in the International Standard Randomized Controlled Trial Number Register with the number ISRCTN31998214.1) was performed by a spirometer, as per American Thoracic Society standards [Patients were randomized to receive their initial treatment based on either the SINA approach  between baseline visit and follow-up visit was done by calculating the Pearson correlation coefficient. A p-value less than or equal to 0.05 was accepted as statistically significant. All randomized patients were included in the analysis, as per the intention-to-treat principle.Sample size calculation, we estimated the controlled patients to be 30% and thus, a sample size of 45 is needed in each arm to have an 80% power to detect a difference of 30%, at an alpha level of 0.05. The data were entered into a Microsoft Excel spreadsheet, which was then transferred into the Statistical Package for Social Sciences (SPSS) program, which was used for data cleaning, management, and analyses. The difference in outcome between the first visit and the follow-up visit was calculated, and then patients with a significant response were flagged. Descriptive analyses were carried out by calculating the number and percent for categorical variables and mean and standard deviation for continuous data. The inferential statistics for the comparison between the two groups was carried out using the Chi-square test for categorical variables, and the 1 values were lower in the SINA group than the GINA group, this difference did not reach statistical significance. Table 1 score of the SINA group (5.8%) than the GINA group (3.4%), this difference did not reach statistical significance (p = 0.46). In contrast, the improvement in PEF was higher in the GINA group compared to the SINA group, but this difference also did not reach statistical significance (p = 0.803). The improvement in the ACT score after treatment initiation was significantly higher in the SINA group compared to the GINA group (2.9 compared to 1.7 (p = 0.04)).Ninety-eight patients were recruited for this study Figure . Forty-fp = 0.185). The correlation between the difference in FEV1 and that of the ACT scores between the initial and follow-up visits was found to be statistically significant, with a correlation coefficient of 0.21 (p = 0.05). Although there was a positive correlation between the changes in FEV1 and PFM (correlation coefficient = 0.19), the change was not significant (p = 0.07). Finally, there was a very weak positive correlation between the changes in the PFM and ACT .Although both groups showed similarities in categorization based on the initial ACT scores table , the folp = 0.0125). Eight (17.8%) patients in the SINA group required a step down in therapy, and 4 required a step up in treatment at the follow-up visit. In comparison, 18 patients in the GINA group required a step down, while no patients required a step up.Figure This study showed that the ACT was responsive to changes at the initiation of asthma treatment . It has p = 0.04). Though the improvement in FEV1 was better in the patients who followed the SINA approach, this difference did not reach statistical significance. This lack of statistical significance could be related to an inadequate sample size to assess a positive response of this outcome. The conflicting results obtained regarding the improvement in PEF in the GINA group may be related to the poor correlation of PEF with the ACT and pulmonary function when used to assess asthma control [1. Moreover, the change in the ACT between the two visits was significantly correlated with the change in the FEV1 and FeNO in that study [1 is the main objective physiological measurement of asthma control, the ACT was also found to correlate well with lung function and inflammation [Utilizing the initial ACT score to determine the appropriate treatment in this study has led to an improvement of 2.9 units, better than the 1.7 unit improvement observed when treatment was determined by physician judgment based on the GINA approach . This was supported by the fact that 40% of those treated with the GINA approach required a step down in treatment upon follow-up, compared to 17.8% of those who treated with the SINA approach. Commencing treatment with the appropriate dose would enhance compliance and minimize the side effects of medications. On the other hand, 8.9% of the patients who followed the SINA required a step-up in treatment compared to none in the GINA group, a finding that may indicate an inadequate initial treatment.Finally, it is worth mentioning a few limitations and concerns related to this study. The utilization of the ACT as an objective measure for initiating asthma therapy is independent of the practitioners' clinical judgment. In contrast, the knowledge of those practitioners who utilized the GINA approach in this study may have been augmented by the pre-study workshop, possibly contaminating the results in that group. This issue was discussed during the preparation of the study protocol, and the authors felt that it was inappropriate to deny the practitioners' placement in the GINA arm due to ethical considerations. In day-to-day practice, most practitioners have variability in their knowledge and experience and may not have the opportunity for dedicated education sessions. Therefore, it is an area for future research to challenge our findings in general practice. Another limitation was the use of set ACT score limits of 16 and 19 for decisions regarding the appropriate initial treatment step. Due to the lack of evidence identifying ACT reference scores for treatment initiation, these numbers were extrapolated from studies that assessed asthma control to make decisions about treatment adjustment and maintenance. Defining the ACT categories that determine initial treatment is another area that needs to be challenged to support its suitability ,27.We believe that despite the aforementioned limitations and concerns, this study can be considered a pilot project that showed the ACT to be responsive to change at treatment initiation and showed usefulness for the initiation of asthma treatment compared to the GINA approach.1: Forced expiratory volumes in one second; FeNO: Fractional exhaled nitric oxide.ACT: Asthma control test; SINA: Saudi Initiative for Asthma; GINA: Global Initiative for Asthma; NAEPP: National Asthma Education and Prevention Program; PEF: Peak expiratory flow; FEVThe authors declare that they have no competing interests.MSAM: primary author, study design, analysis, and writing of the manuscript. MOAG: study design, data collection, and writing of the manuscript. AGAK: data collection, approvals, and writing of the manuscript. HMT: study design, randomization, data analysis and manuscript writing. All authors read and approved the final manuscript.This study was supported by a grant from the King Abdullah International Center for Medical Research.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/12/14/prepub


Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data  separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray,  Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen, is the causative agent for human and animal listeriosis. The invasive form of human listeriosis (Inv) is a disease predominantly affecting immuno-compromised people, the elderly, neonates and pregnant women and is characterized by septicemia and/or meningitis in non-pregnancy-associated cases, fetal loss, premature labor and neonatal septicemia or meningitis in pregnancy-associated cases with high case-fatality ratios (∼20–30%) L. monocytogenes is not routinely cultured from stool in clinical laboratories.L. monocytogenesAlthough the majority of the large listeriosis outbreaks detected in the United States have been associated with the consumption of ready-to-eat frankfurters and deli meats, as well as milk and dairy products, outbreaks due to consumption of produce  also have been previously reported L. monocytogenes were obtained from samples of blood or cerebrospinal fluid. The outbreak strains consisted of serotypes 1/2a and 1/2b and belonged to four distinct pulsed-field gel electrophoresis (PFGE) pattern combinations (PCs) I, II, III, and IV. Investigation by local, state and federal public health and regulatory agencies identified L. monocytogenes outbreak strains on cantaloupes collected from grocery stores, ill person's home, and whole cantaloupes collected from cold storage and the packing facility environment; environmental swabs collected at the facility also identified L. monocytogenes outbreak strains L. monocytogenes in the cantaloupe From August 12 to November 1, 2011, 146 outbreak-associated cases of invasive listeriosis were diagnosed among residents of 28 states in the USA. Thirty deaths and one miscarriage were reported during the outbreak, which was the largest number of fatalities due to an outbreak of foodborne listeriosis in the United States L. monocytogenes can be classified into 13 serotypes Listeria GeneChip® L. monocytogenes genomes available in public databases as of 2009. Using Listeria GeneChip®, we have already shown that the outbreak strains of L. monocytogenes can be further classified into different genomic groups or genovars and it could also identify epidemic clones and could further distinguish the individual outbreak strains and food and clinical isolates Although L. monocytogenes isolates were identified using methods described in the Bacteriological Analytical Manual L. monocytogenes isolates were typed following the standard PulseNet procedure using restriction enzymes AscI and ApaI  Cantaloupe outbreak isolates  were gro+), 20 mM EDTA, 0.01% Tween-20, 50 pM control oligoB2 , 0.1 mg/ml herring sperm DNA (Promega), 7.8% dimethylsulfoxide (DMSO) , were heated at 95°C for 1 minute followed by incubation at 45°C for 5 minutes, prior to hybridizing onto the Affymetrix Listeria GeneChip® at 45°C with rotation (60 rpm) for 16 hours in a hybridization oven. The wash and staining procedures were carried out on an Affymetrix FS-450 fluidics station using the mini_prok2v1_450 fluidics script as described by GeneChip® Expression Analysis Technical Manual with the slight modification previously described Hybridizations of the genomic DNA isolated from cantaloupe outbreak strains were performed according to the Affymetrix GeneChip® Expression Analysis Technical Manual All Affymetrix CEL files generated in this study were parsed and analyzed using algorithms including MAS5.0 L. monocytogenes serotype 1/2a and 1/2b was constructed using the uncorrected p-distance in Splitstree 4.11.3.The gene present/absent binary nucleotide calls based on the gene contents as T (present) and A (absent), were concatenated for each strain, such that a 18,630 bp sequence was generated to represent the gene content L. monocytogenes. Although the majority of recorded invasive listeriosis outbreaks were caused by serotype 4b, serotype 1/2a and 1/2b have been reported more frequently in recent years. Serotype 1/2a and 1/2b isolates have also been involved in several FG listeriosis outbreaks All isolates showed a positive agglutination reaction with Difco Type 1 antisera. Combining these results with the multiplex PCR results (data not shown) allowed us to make a final serotype determination within 24 hours of inoculating the initial BHI-A overnight culture. The multiplex, PCR-based serotypic determinations of several strains were independently confirmed by a standard antisera-based serotyping protocol using the Denka-Seiken commercial kit (data not shown). Thus, the multiplex PCR-agglutination protocol allowed us to rapidly and accurately determine the serotypes for the outbreak isolates. These analyses confirmed that this outbreak was a multi-strain and multi-serotype outbreak, involving two distantly related serotypes Molecular subtyping is one of the crucial tools for outbreak investigation. Since 2001 standardized PFGE has become the gold standard for establishing genetic relatedness among bacterial isolates obtained from foods, clinical samples and the environment AscI and ApaI) from selected outbreak strains ; however, the AscI patterns  differed by a band shift of ∼40 kb possibly due to the presence of a phage in PC IV isolates . The PCs assigned to each isolate examined are shown in PFGE patterns  of these PCs in the database ranged from 6 to 60 with most of these isolates belonging to PC II . InteresIt is clear from the above paragraphs that the cantaloupe outbreak was caused by at least three strains belonging to two different serotypes. Four PCs were identified, of which two were closely related. In order to further characterize these strains at the genomic level, we chose eight isolates from food or the food processing environment and eight isolates from patients for microarray analysis .L. monocytogenes strains were identified from the microarray results using the Affy package in R-Bioconductor. The gene contents of the cantaloupe outbreak strains from both serotypes 1/2a and 1/2b were compared with the unrelated strains, which are ones we have previously characterized Listeria GeneChip®, 7,823 probe-sets were conserved, which left 4,041 (21.7%) and 6,766 probe-sets (36.3%) as present and absent, respectively. A neighbor-net tree, constructed based on the gene content analysis, clearly separated these cantaloupe outbreak and unrelated strains according to their serotypes (The gene contents (present/absent) of these erotypes . Previouerotypes  were fouL. monocytogenes isolates serotype 1/2a implicated in this 2011 cantaloupe outbreak, the PC I strains were genetically distant from the other two PCs, which in turn are closely related, confirming the PFGE results. Further comparison of genetic contents in the PCs III and IV with those from PC I revealed that approximately 3% of probe-sets were uniquely present in each group  using MAS5.0 algorithm. Gene content comparison among these cantaloupe outbreak isolates, an unmatched shrimp isolate (LS693), and previously reported strains ch group  and S2. To further analyze the genetic variability of the serotype 1/2a isolates, Robust Multi Array (RMA) algorithm L. monocytogenes serotype 1/2a strains were most amenable to phage transduction compared to other serotypes, including 1/2b and 4b comK could provide an added advantage for Listeria to survive in selective environments comK site. Thus, the increased ability to accept, maintain, and recombine/mutate phage gene sequences in serotype 1/2a PC IV strains could potentially contribute to the evolution of a more fit L. monocytogenes genovar. This raises an important question about the potential risk associated with the using of bacteriophages to control Listeria in foods. However, experimental evidence is required to determine whether isolates harboring the phage have a selective advantage relative to those that do not carry the phage and under what conditions the phage confers such an advantage.The majority of the serotype 1/2a isolates from the cantaloupe outbreak strains analyzed here belong to the PCs III and IV. Comparison of gene contents from all of these six isolates based on MAS5.0 analysis showed that 7,643 probe-sets were present and 10,415 probe-sets were absent, which encompassed 97% of conserved probe-sets. This result indicated that isolates from the PCs III and IV are more closely related to each other than to the PC I strains and could be considered variants of the same strain. There were three unique probe-sets present in the isolates from PC III, including two hypothetical protein genes and one intergenic region. On the other hand, the PC IV strains contained an additional 110 unique probe-sets , Fig. 8 The genetic relationship of the cantaloupe outbreak serotype 1/2b strains was examined based on the gene contents information obtained from the microarray analysis. The results indicated that 87.9% of total probe-sets were conserved among 1/2b isolates included in this study. Although the isolates from the cantaloupe outbreak are clustered together , the genAfter these analyses were completed, a state public health laboratory retrospectively identified a strain with a PFGE pattern combination different from all other PC in the national data base. This strain was detected on a sample of cantaloupe and only a single case of human illness was reported during the outbreak.L. monocytogenes isolates that caused the large, multistate outbreak of listeriosis in 2011. The outbreak was unique in the sense that it was the first documentation of a listeriosis outbreak associated with consumption of contaminated cantaloupe in the United States and was caused by several distinct serotypes and genotypes of L. monocytogenes. The increasing discriminatory powers of the three analytical tools applied in this study underscores the utility of multiphasic approach in revealing the complex polyclonal nature of this outbreak. Our microarray analysis showed that the 1/2a and 1/2b strains associated with this outbreak are distinctly different from the previously reported 1/2a and 1/2b strains associated with both Inv and FG listeriosis outbreaks and sporadic cases. The pan-genomic analysis of these strains provided a powerful tool for exploring the genomic footprints of these organisms and underlined the possibility that phage genomes may be an important driving force behind the evolution of some strains of L. monocytogenes.To summarize, a combination of serotyping, PFGE and DNA microarray hybridization analyses were employed to characterize the genomic footprints of the Table S1Probe-sets uniquely present in PC I serotype 1/2a.(DOCX)Click here for additional data file.Table S2Probe-sets uniquely present in PC III and IV strains serotype 1/2a.(DOCX)Click here for additional data file.Table S3Probe-sets uniquely present in PC IV serotype 1/2a.1.(DOCX)Click here for additional data file.


Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism. Listeria monocytogenes is a Gram-positive foodborne bacterial pathogen responsible for human and animal listeriosis. Recent data L. monocytogenes have been reported L. monocytogenes is not known because FG cases are not routinely screened for L. monocytogenesL. monocytogenes strains can be classified into 13 serotypes L. monocytogenesL. monocytogenes strains isolated from these outbreaks. Franciosa et al (2001) analyzed a total of 32 strains, 16 from Inv and 16 from FG listeriosis outbreaks by ribotyping, arbitrarily primed PCR (AP-PCR) and interspersed repetitive sequence PCR (IRS-PCR) L. monocytogenes serotype 4b and 1/2b strains from Inv and FG listeriosis by several other molecular sub-typing techniques Based on somatic and flagellar antigens, L. monocytogenes appear to be highly clonal. These molecular subtyping methods revealed that L. monocytogenes can be classified into at least three lineages correlated to their serotypes L. monocytogenes strains associated with different outbreaks using molecular subtyping methods divide these strains into five epidemic clones (ECs), suggesting that strains causing major outbreaks are genetically related Based on several molecular subtyping studies including multi-locus enzyme electrophoresis (MLEE), RFLP and PFGE, genetic structures of L. monocytogenesL. monocytogenesL. monocytogenes based on the publicly available information (as of May 2009) of 24 L. monocytogenes genome sequences. Using our custom Listeria GeneChip (Affymetrix technology), we analyzed 38 L. monocytogenes strains isolated from Inv and FG listeriosis outbreaks. The strains represent serotypes 1/2a, 1/2b and 4b including epidemiologically matched clinical and food isolates. Our results show that the microarray-based analysis using this GeneChip can be used as an outbreak investigation tool to identify genome differences and separate L. monocytogenes strains based on their serotype, epidemic clone type and outbreaks. The distinct difference in genetic footprints between strains of FG and Inv outbreaks may help in understanding the diverse pathophysiology of this organism.With the advent of whole genome sequencing technology and the availability of advanced bioinformatics tools, it is possible to identify small changes in the genetic makeup of bacterial pathogens, including L. monocytogenes were obtained from various sources . Briefly, 200µl hybridization reactions containing 10µg of labeled fragmented DNA, 100mM MES, 1M(Na+), 20mM EDTA, 0.01% Tween-20, 50pM control oligoB2 , 0.1 mg/ml herring sperm DNA (Promega), 7.8% dimethylsulfoxide (DMSO) , were heated at 95°C for 1 minute followed by incubation at 45°C for 5 minutes, prior to hybridizing onto the Affymetrix Listeria GeneChip at 45°C with rotation (60rpm) for 16 hours in a hybridization oven. The buffer preparation, the wash and staining procedures were carried out on an Affymetrix FS-450 fluidics station using the mini_prok2v1_450 fluidics script as described by GeneChip Expression Analysis Technical Manual with the slight modification that Streptavidin solution mix was replaced with Streptavidin, R-phycoerythrin conjugate (SAPE) . Arrays were subsequently scanned using a GeneChip Scanner 3000 7G with GCOS v1.4 software.Hybridizations were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual (L. monocytogenes microarray (Listeria GeneChip) is custom designed using Affymetrix chip technology and has components for use as an expression or genotyping array and probes for use as a tiling array. The Listeria expression/genotyping microarray was designed to represent 64,539 annotated gene sequences from 24 sequenced strains of L. monocytogenes which were available from GenBank and Broad Institute (http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html) as of May, 2009 and 7,354 intergenic sequences from four of the sequenced L. monocytogenes strains  (The ip81459) . IdenticThe tiling portion of the array consists of 568,677 probes covering the whole genome of AE017262 4b F2365. The 25-mer probes cover the genome at 4-nt gaps between starts, allowing for 20-nt overlap between probes. Each nucleotide in the genome is covered by 5 probes for detection of Single Nucleotide Polymorphisms (SNPs) relative to the reference sequence.All Affymetrix CEL files generated in this study were parsed and analyzed using algorithms including MAS5.0 L. monocytogenes gene contents for which the presence or absence of genes were coded as T (present) or A (absent), respectively. The gene present/absent binary nucleotide calls were concatenated for each strain, such that a 18,630 bp sequence was generated to represent the gene content. Genes that are not phylogenetically informative L. monocytogenes serotype 1/2a, 1/2b and 4b was constructed using the uncorrected p-distance in Splitstree 4.11.3.Affymetrix MAS5.0 algorithm using Affy package of R and Bioconductor was used to identify L. monocytogenes genome sequences allowed us to design a GeneChip that integrates sequences from many genomes in one single GeneChip. In this study, we investigated the L. monocytogenes genome diversity using Affymetrix high-density microarray GeneChip that was custom designed based on 24 L. monocytogenes genome sequences available at the time of GeneChip design (Listeria GeneChip was designed to study genome diversity and gene expression as well as single nucleotide polymorphisms (SNPs) of L. monocytogenes. All probe-sets on Our Listeria GeneChip consist of up to 14 probe-pairs per gene, phage gene and intergenic region. Each probe-pair contains one perfect-match probe (PM) and another with one nucleotide mismatch (MM). This feature enables us to perform highly accurate gene content detection.The availability of p design  .The LisL. monocytogenes strains, MAS5.0 gene detection approach was used p-values. In addition to the R Score, sensitivity and/or specificity of gene detection depends on a small positive threshold value, Taup-value using a one-sided Wilcoxon Signed Rank test as described by Jackson et al. p-value <0.05 were scored as present and ≥0.05 as absent. Tau values on gene present calls for strains LS402, LS406 and LS411. Increased Tau values clearly resulted in reduced numbers of gene present calls that correspond to false negatives. However, higher Tau values will also result in reduced numbers of truly present genes . In addition, it is important to note that our Listeria GeneChip was designed based on the available genome sequences to study global genomic diversity. Several probe sets may contain probes that share different percent identities from the same genes in various genomes. The predicted numbers of present genes, therefore, can or often do exceed the true numbers of genes in the L. monocytogenes genomes, providing better resolution for gene detection (To determine gene contents (present/absent) in etection .Tau values to the individual CEL files in the triplicate experiments, we found that the data reproducibility changed, depending largely on the Tau value selection. Tau values provided results with lower percent reproducibility. Tau values up to 0.3, on the other hand, increased the percent reproducibility to approximately 98%. While increasing Tau values above 0.3 resulted in reduced percent reproducibility, Tau values above 0.5 again raised the percent reproducibility. This increase in reproducibility resulted from more false absent calls occurring with Tau values above 0.5, . As a result, 10,592 out of 18,630 total probe-sets were automatically scored as absent. Although 8,038 probe-sets may be called as present, we found that 6,980 probe-sets can be scored as present based on the following two criteria. First, each probe-set must contain at least 40% of 100% matched probes compared to the total probe numbers. Secondly, each probe-set screened by the first criteria must contain at least six 100% matched probes, allowing at least 150 nucleotides to be detected. The numbers of nucleotides are therefore sufficient for gene detection. The gene detection calls from the triplicate LS411 hybridization experiments using varied Tau values were then compared with the gene present/absent calls from the BLAST analysis to identify the numbers of false positive and false negative. As expected, with increasing Tau values, the numbers of false negative rose due to more absent calls, whereas the numbers of false positive dropped exponentially . This analysis strongly indicated that Tau values between 0.2 and 0.3 provide the most accurate gene detection calls for the Listeria GeneChip hence the Tau value of 0.25 was subsequently used in the downstream analyses to identify the gene contents of different L. monocytogenes strains. A similar study using Affymetrix GeneChip® E. coli Genome 2.0 revealed that a Tau value of 0.2 provided the most accurate gene present/absent calls To further validate our e LS411,  [19], thentially . FurtherListeria GeneChip by comparing summarized probe-set intensities, independent to MAS5.0 algorithm. In contrast to MAS5.0 gene present/absent call analysis, MM probes are not considered as a part of the RMA calculation. The summarized probe-set intensities were therefore determined based on PM probes alone. As a part of the result validation, the same CEL files used in the MAS5.0 analysis from the triplicate hybridization experiments performed in some strains were subjected to RMA analysis using Affy package in R-Bioconductor. The summarized probe intensities among triplicate experiments with LS402, LS406 and LS411 were compared. The RMA scatter plots between the samples in triplicate (Listeria GeneChip is improved resolution as shown in The Robust Multi-array Averaging (RMA) approach iplicate  A–F reveiplicate  by scattiplicate  whereas iplicate . This reiplicate  A–C, theL. monocytogenes strains into 2 clusters correlating to their ECs (ECI and IV) while they are clearly distinct from the serotype 1/2b strains. The heat map reveals distinct trends in the differences of gene content among strains from different ECs as also established by previous study in E. coliWe further validated the microarray results using the RMA-summarized probe intensities generated from serotype 4b (ECI and ECIV) and serotype 1/2b strains. A heat map generated from the RMA-summarized probe intensities shows that, when the individual 4b strains were examined in triplicate, the results appeared to be identical suggesting consistency in the array data . The denL. monocytogenes strains, genomic content information (present/absent) was analyzed using MAS5.0 algorithm to infer strain relatedness. All CEL files generated from the genomic DNA hybridization of the 38 L. monocytogenes strains from the three serotypes  were parsed and analyzed using Affy package in R-Bioconductor with the Tau value of 0.25. There are 18,360 probe-sets on the array, of which 8,079 probe-sets are conserved (either present or absent) across all 38 strains , likely representing a large component of the core L. monocytogenes genome. As a result, they are not phylogenetically informative and were consequently excluded from the analysis L. monocytogenes. The 9,767 parsimoniously informative sites were selected from the 10,551 phylogenetically informative probe-sets. A neighbor-joining tree of the 38 strains constructed using the gene content information, separated the strains into their respective serotypes and epidemic clones . The serotype 4b strains were divided into four distinct clusters corresponding to their ECs . Interestingly, the parallelogram analysis revealed that there may be more substantial mutations or recombinations in ECI, ECII and ECV than those of the ECIV and serotype 1/2b strains. Pairwise homoplasy index (PHI) p-value of 0.0 which confirms that there was significant evidence of recombination or parallel gene gain/loss due to multiple transduction events. In addition, when 925 phage genes were removed from the analysis, the topology and clusters of the resulting tree were unaffected (data not shown) suggesting the stability of the genomes and the relationship among these L. monocytogenes strains has not been influenced by phages.The concatenated sequences of the probe-sets (present/absent) were also examined using Neighbor-net in Splitstree program L. monocytogenes genomes, is unique in 10 strains belonging to the ECI Currently, serotype 4b strains are divided into four ECs in which each of them are harboring unique probe-sets , S7, S8.L. monocytogenes genomes are very similar (syntenic) and most of the differences are due to phage genomes and transposable elements as well as SNPs L. monocytogenes may not require many genetic elements to adapt to different environments and exhibit different virulence attributes as suggested by Nelson et al.comK prophage in L. monocytogenes for niche-specific adaptation, biofilm formation and persistence has been recently demonstrated L. monocytogenes.Previous study involving the whole genome comparison between the serotypes 1/2a and 4b strains revealed that Based on the genomic profiles of 31 different strains representing serotypes 1/2b and 4b , both FG and Inv listeriosis strains from matched pairs of food and clinical isolates are more similar closely related than to those from the different outbreaks . HoweverL. monocytogenes genomes from the public databases and genomic analysis of L. monocytogenes outbreak strains using this GeneChip. Gene detection methods using the MAS5.0 algorithm to identify gene presence and absence have been optimized for our GeneChip based on known sequences. The numbers of present genes called may exceed the true numbers of genes in L. monocytogenes genomes due to redundancy of probes within probe-sets as a result of our microarray design in order to study global diversity of L. monocytogenes genomes. We showed that the results obtained from either RMA or MAS5.0 approaches are consistent, suggesting the reliability and validity of the data. The gene content analysis using the phylogenetically informative sites revealed that L. monocytogenes strains are divided into three distinct groups correlating with the serotypes. Strains belonging to the serotype 1/2a are more genetically distant from those of 1/2b and 4b strains. Within the same serotype, strains that belong to the same ECs are clustered closely together. Comparison of the serotype 4b, ECIV FG and Inv strains indicated that the majority of the uniquely present probe-sets in FG isolates are phage-related genes suggesting that phages may play a significant role in the divergence of these two pathotypes and may play important roles in pathotype determination. We showed that our high density microarray can identify genetic contents that are specific to serotypes, pathotypes and epidemic clones. Our results also indicated that microarray based genotypic analysis can be a very important tool in outbreak investigation as closely related members of the same serotype as well as the food and clinical isolates derived from outbreaks can further be differentiated from each other.In conclusion, we report the design of a microarray GeneChip consisting of 24 Table S1Listeria monocytogenes strains used in this study.(DOCX)Click here for additional data file.Table S2Probe-sets uniquely present in the serotype 1/2a strains.(DOCX)Click here for additional data file.Table S3Probe-sets uniquely present in the serotype 1/2b strains.(DOCX)Click here for additional data file.Table S4Probe-sets uniquely present in the serotype 4b strains.(DOCX)Click here for additional data file.Table S5Probe-sets uniquely present in the serotype 4b, epidemic clone I.(DOCX)Click here for additional data file.Table S6Probe-sets uniquely present in the serotype 4b, epidemic clone II.(DOCX)Click here for additional data file.Table S7Probe-sets uniquely present in the serotype 4b, epidemic clone IV.(DOCX)Click here for additional data file.Table S8Probe-sets uniquely present in the serotype 4b, epidemic clone V.(DOCX)Click here for additional data file.Table S9Probe-sets uniquely present in the serotype 1/2b strains that cause invasive listeriosis.(DOCX)Click here for additional data file.Table S10Probe-sets uniquely present in the serotype 1/2b strains that cause febrile gastroenteritis listeriosis.(DOCX)Click here for additional data file.Table S11Probe-sets uniquely present in the serotype 4b, ECIV strains that cause invasive listeriosis.(DOCX)Click here for additional data file.Table S12Probe-sets uniquely present in the serotype 4b, ECIV strains that cause febrile gastroenteritis listeriosis.(DOCX)Click here for additional data file.


Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of  The Food and Drug Administration (FDA) inspected the involved farm; outbreak strains matching 3 of the PFGE profiles from clinical samples were isolated from washed cantaloupes and various environmental surfaces within the facility (www.fda.gov/Food/FoodSafety/CORENetwork/ucm272372.htm#report).In September 2011, the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, was notified of an increase of listeriosis cases linked to eating cantaloupe  sequences were demonstrated to be unique to EC strains of L. monocytogenes in individual facilities that processed ready-to-eat meat and poultry or in multiple plants manufacturing similar ready-to-eat products  of comK prophage JF sequencing   and by FDA according to the FDA Bacteriological Analytical Manual . Isolates were serotyped by using commercial antisera  and analyzed by PFGE (CDC confirmed identification of  by PFGE . The Tecwww.pasteur.fr/mlst). MVLST data were obtained as described  and 3 USDA isolates (accession nos. JQ750615–JQ750618).Isolates were grown overnight in tryptic soy broth with yeast extract at 37°C, and DNA was extracted by using the Ultra Clean Microbial DNA Isolation Kit  for isolates from CDC and USDA and the Wizard Genomic DNA Purification Kit  for isolates from FDA. Sequence types (STs) identified by using MLST were assigned as described  belonged to the globally disseminated ST5 ,. Becausehage JFs . PT11/11hage JFs . FurtherinlC from 3 other serotype 1/2a VT61 isolates  associated with the 2002 cheese-associated listeriosis outbreak in Canada  from cantaloupe differed by 1 single nucleotide polymorphism in  Canada ,. L2625 w,Listeria isolates from cantaloupe displayed 2 highly similar PFGE profiles and STs, and the same serotype, ApaI PFGE pattern, and VT  and L2676, LIS0072, and LIS0087  from cantaloupe samples shared the same VT (VT56) as isolates 10-0813 and 10-0812 associated with a listeriosis outbreak related to whipping cream during 2000 in Canada ,. These L, and VT . Isolaten Canada . The JF n Canada . These in Canada . HoweverL. monocytogenes strains, of the same genetic lineage as serotype 1/2b strains, reportedly survived and grew substantially better in mixed-serotype biofilms containing a specific strain of serotype 1/2a (Different clones, particularly ECVI and ECVII, might have cocolonized niches or harborage sites within the cantaloupe processing facility, possibly explaining the multiple strains associated with this outbreak. Serotype 4b ,L. monocytogenes.Six of the 7 currently identified ECs were found at some point in 1 or both of the US chicken processing plants included in the study . ListeriL. monocytogenes strains involved in the 2011 multistate cantaloupe-associated outbreak was greatly enhanced by the use of subtyping markers with different levels of epidemiologic resolution. Particularly, MVLST enabled the detection of 1 novel 1/2a outbreak strain and 2 novel ECs of L. monocytogenes. In contrast to focusing on isolates from a single outbreak (The molecular epidemiology of Listeria monocytogenes encountered in clinical and food or environment samples collected by the Centers for Disease Control and Prevention during a 2011 L. monocytogenes outbreak related to cantaloupe.Number of isolates of


Background: The development of left and right superior temporal gyrus (STG) 50 ms (M50) and 100 ms (M100) auditory responses in typically developing (TD) children and in children with autism spectrum disorder (ASD) was examined. Reflecting differential development of primary/secondary auditory areas and supporting previous studies, it was hypothesized that whereas left and right M50 STG responses would be observed equally often in younger and older children, left and right M100 STG responses would more often be absent in younger than older children. In ASD, delayed neurodevelopment would be indicated via the observation of a greater proportion of ASD than TD subjects showing missing M100 but not M50 responses in both age groups. Missing M100 responses would be observed primarily in children with ASD with language impairment (ASD + LI) .Methods: Thirty-five TD controls, 63 ASD without language impairment (ASD − LI), and 38 ASD + LI were recruited. Binaural tones were presented. The presence or absence of a STG M50 and M100 was scored. Subjects were grouped into younger (6–10 years old) and older groups (11–15 years old).Results: Although M50 responses were observed equally often in older and younger subjects and equally often in TD and ASD, left and right M50 responses were delayed in ASD − LI and ASD + LI. Group comparisons showed that in younger subjects M100 responses were observed more often in TD than ASD + LI , with no differences between TD and ASD − LI  or between ASD − LI and ASD + LI . In older subjects, whereas no differences were observed between TD and ASD + LI, responses were observed more often in ASD − LI than ASD + LI. Findings were similar when splitting the ASD group into lower- and higher-cognitive functioning groups.Conclusion: Although present in all groups, M50 responses were delayed in ASD. Examining the TD data, findings indicated that by 11 years, a right M100 should be observed in 100% of subjects and a left M100 in 80% of subjects. Thus, by 11 years, lack of a left and especially right M100 offers neurobiological insight into sensory processing that may underlie language or cognitive impairment. Autism spectrum disorders (ASD) are a set of developmental disorders characterized by social impairments, stereotypical behaviors, and deficits in communication. As a childhood disorder, an understanding of brain abnormalities in ASD requires an examination of brain processes in infants, toddlers, and young and older school-aged children with ASD. A growing number of electroencephalography (EEG) and magnetoencephalography (MEG) studies report auditory abnormalities in children with ASD. Findings include delayed superior temporal gyrus (STG) auditory 100 ms responses in children with ASD  children  and M100 (MEG) are the most prominent deflections of the auditory event-related potential (EEG) or field (MEG), evolving with a peak latency of about 100 ms after stimulus onset  is also often seen. The relevant MEG literature points to STG as the M50 generator  peak latency in children 5–6 years of age ~85–95 ms  .Diagnoses of ASD were determined prior to recruitment based on the child’s performance during clinical interviews, their documentation of DSM-IV criteria for ASD, and results from tests such as the Childhood Autism Rating Scale and the autism diagnostic observation schedule (ADOS). Advertisements through local newspapers and pediatric practices within the Children’s Hospital of Philadelphia (CHOP) primary care network were utilized for recruitment of TD controls.The subjects’ first session at CHOP included clinical and diagnostic neuropsychological testing by a licensed child psychologist with expertise in autism (Lisa Blaskey) to ensure that all subjects met the minimum criteria for inclusion and to further confirm diagnoses of ASD, particularly by utilizing the ADOS, Social Responsiveness Scale (SRS), Krug Asperger’s Disorder Index (KADI), and Social Communication Questionnaire (SCQ). For confirmation of the ASD diagnosis, all children had to exceed established cut-offs on both the ADOS and SCQ. Subjects one point below cut-off for ADOS scores were permitted entry into the study as long as they exceeded cut-offs on at least two parent questionnaires. In the rare event that diagnosis could not be confirmed via use of the ADOS and parent questionnaires alone, the autism diagnostic interview-revised (ADI-R) was administered to provide final clarification of diagnosis.For testing language impairment, all subjects were evaluated with the clinical evaluation of language fundamentals – fourth edition (CELF-4). The ASD sample was divided into ASD without language impairment (ASD − LI) and ASD + LI groups. The ASD + LI group included subjects scoring at or below the 16th percentile (SS < 85) on the CELF-4 Core Language Index. All subjects scored at or above the 5th percentile (SS > 75) on the perceptual reasoning index (PRI) of the Wechsler Intelligence Scale for Children-IV (WISC-IV). The WISC-IV verbal comprehension index (VCI) was also obtained.The total number of subjects included 35 TD and 101 ASD . Although analyzing data from a different task, the present sample includes several, although not all, of the subjects reported in Roberts et al.  , an 800 ms ISI, a second 50 ms tone , and a 2350 ms (±100) inter-trial interval. A total of 120 tone pairs were presented. The present study reports on S1 – the auditory response occurring after the long inter-trial interval and thus with the greatest recovery period. To obtain a response with sufficient trials, the S1 average included 1000 and 2000 Hz tones.Stimuli consisted of 1000 and 2000 Hz tones presented using Eprime v1.1. Tones were presented via a sound pressure transducer and sound conduction tubing to the subject’s peripheral auditory canal via ear-tip inserts . Prior to data acquisition, 1000 Hz tones of 300 ms duration and 10 ms rise time were presented binaurally and incrementally until reaching auditory threshold for each ear. Tones were presented at 45 dB sensation level above threshold. Each trial consisted of a 50 ms tone  in a magnetically shielded room. Prior to data acquisition, three head-position indicator coils were attached to the subject’s scalp at the nasion, left-, and right-preauricular points, providing continuous specification on head position and orientation in relation to the MEG sensors. A movie (without sound) was displayed to prevent fatigue.Electrodes were attached to the left and right clavicles for electrocardiogram recordings (ECG) and to the bipolar oblique (upper and lower left sites) for electro-oculogram recordings (EOG). A band-pass filter (0.03–150 Hz) was placed over the EOG, ECG, and MEG signals, which were then digitized at 1200 Hz with third order gradiometer environmental noise reduction over the MEG data.F = 8.82, p < 0.001, the difference between groups in the mean number of trials was small: TD mean of 110 trials (range 83–119) = ASD − LI mean of 107 trials (range 86–119) > ASD + LI mean = 101 trials (range 81–116).Artifact correction was applied to remove eye-blink and cardiac activity  using a model with multiple sources . In particular, a M100 was scored if the magnetic flux topography were characteristic of the M100 response, was preceded by M50 , and followed by M200 , and with source strength greater than baseline. In the present study, M50 was operationally defined as the first reversal in magnetic-field topography preceding M100 (or M200 if M100 not present). As reported below, in many subjects, a left- or right-hemisphere M100 response was not observed. For these subjects, left and right STG dipoles were oriented at the maximum of M50. If neither a M50 nor M100 was observed, the dipole was oriented at M200. Identification of auditory responses and orientation of the STG dipoles were done blind to group.p > 0.05]. Examining all subjects, maximum head motion during the recording was greater in younger than older subjects .Of the subjects examined, 18 subjects  did not have observable M50, M100, or M200 responses in the left- or right-hemisphere. Lack of an observable auditory response in these subjects was due to large metal artifact or poor compliance. Data from these subjects was excluded. Examining only the subjects with an identifiable auditory response, goodness-of-fit values (average from the start of M50 to M200) did not differ between the TD and ASD groups [TD = 94% (SD = 2.28), ASD = 93% (SD = 3.00); In subjects with usable data , epochs were defined from the continuous recording: 500 ms before the first tone to 500 ms after the first tone. When a M50 or M100 response was observed, left and right M50 (35–120 ms) and M100 (80–185 ms) latency data were calculated from the largest point in the scoring windows using in-house MATLAB software after subtraction of prestimulus baseline activity. Given that in many subjects the M200 was of long duration and without a clear peak, M200 responses were simply scored as present or absent, with M200 defined as a sustained response occurring after the M100 interval  and showing a magnetic-field topography similar to M100.t-tests examined group differences in age, CELF-4 scores, SRS, and IQ scores. For between-subject analyses, chi-square tests examined group differences in the presence or absence of a M50, M100, and M200 response . For within-subject analyses, McNemar tests were used , hemisphere, and group × hemisphere differences in latency. To examine how M50 and M100 latency differs as a function of group and age, hierarchical regression was run entering age first, diagnosis second, and their interaction last, with M50 or M100 latency as the dependent variable.As shown in Table p = 0.27). Collapsing across group and age, the presence or absence of M50 differed between the right (85.4%) and left-hemisphere . Given a significant difference between hemisphere but not age, simple effect analyses examined group differences for each hemisphere, collapsing across age group.Collapsing across group and hemisphere, M50 responses were observed equally often in older (88.3%) and younger subjects , TD (91.7%) and ASD − LI , or ASD − LI (96.8%) and ASD + LI . In the right-hemisphere, collapsing across age, no differences were observed between TD (88.9%) and ASD + LI , TD (88.9%) and ASD − LI , or ASD − LI (85.7%) and ASD + LI . Splitting at the median head motion value, within each diagnostic age group, chi-square analyses showed that the presence or absence of M50 did not differ between individuals with more versus less head motion.In the left-hemisphere, collapsing across age, no differences were observed between TD (91.7%) and ASD + LI . Collapsing across group and age, the presence or absence of M100 did not differ between the right (85%) and left-hemisphere . Given a significant difference between age groups but no hemisphere difference, simple effect analyses examined group differences for each age group collapsing across hemisphere.Collapsing across group and hemisphere, M100 responses were observed more often in older (89.4%) than younger subjects . No differences were observed between TD (90%) versus ASD − LI  or between ASD − LI (76%) and ASD + LI . In older subjects, collapsing across hemisphere, whereas no differences were observed between TD (91%) and ASD + LI , or between TD (91%) and ASD − LI , M100 responses were observed more often in ASD − LI (95%) than ASD + LI . Given similar differences in the presence/absence of M100 between TD versus ASD + LI and ASD − LI versus ASD + LI, the non-significant TD versus ASD + LI finding is likely due to a smaller N in the TD group. Splitting at the median head motion value, within each diagnostic age group, chi-square analyses showed that the presence or absence of M100 did not differ between individuals with more versus less head motion.In younger subjects, collapsing across hemisphere, M100 responses were observed more often in TD (90%) than ASD + LI . Marginally significant differences were observed between TD (88%) versus PRI-low . No differences were observed between PRI-high (78.2%) and PRI-low . In older subjects, collapsing across hemisphere, no differences were observed between TD (91%) and PRI-high , between TD (91%) and PRI-low , or between PRI-high (91.3%) and PRI-low . Although not exactly the same, findings splitting children with ASD into low and high PRI groups were similar to findings splitting children with ASD into low and high CELF-4 groups. This is likely due to the fact that group membership remained largely unchanged. In particular, of the 25 ASD + LI subjects, 19 were in the lower half and 6 in the upper half of the PRI group, and of the 48 ASD − LI subjects, 9 were in the lower half and 39 in the upper half of PRI group.To assess if any of the missing M100 findings were specific to language impairment, the above analyses were re-run dividing the children with ASD into low and high PRI groups (median split). In younger subjects, collapsing across hemisphere, M100 responses were observed equally often in TD (88%) and PRI-high . Collapsing across group and age, the presence or absence of M200 did not differ between the right (96.4%) and left-hemisphere . Given no significant differences between age or hemisphere, simple effect analyses examined group differences for each group collapsing across age and hemisphere.Collapsing across group and hemisphere, M200 responses were observed equally often in older (94.7%) and younger subjects , TD (97.2%) and ASD − LI , or ASD − LI (98.4%) and ASD + LI .No differences were observed between TD (97.2%) and ASD + LI  = 4.59, p = 0.01, showed marginally earlier responses in TD (67 ms) versus ASD − LI  and significantly earlier responses in TD versus ASD + LI . The ASD groups did not differ (p = 0.74). The main effect of hemisphere, F = 0.11, p = 0.74, and the hemisphere by group interaction, F = 1.41, p = 0.25, were not significant. Findings were unchanged re-running analyses with age as a covariate. The pattern of findings was unchanged splitting the ASD group based on PRI, with the earliest M50 latencies in TD, second earliest in the PRI-high group, and the longest in the PRI-low group (PRI-low and PRI-high groups did not differ).ANOVAs examined hemisphere and group latency differences in the subjects with a M50 and M100 response. For M50, simple effect analyses of a main effect of group, F = 14.03, p < 0.001, showed earlier responses in the right (119 ms) than left (126 ms). There was no main effect of group, F = 1.42, p = 0.24. Although the interaction term was not significant, given the right-hemisphere TD versus ASD group latency findings in Roberts et al.  = 1.06, p = 0.31, a significant group M100 latency difference observed in the right-hemisphere, F = 4.87, p = 0.03, indicated earlier M100 latencies in TD  versus ASD . Findings were unchanged re-running analyses with age as a covariate. Analyses also showed M100 right-hemisphere latency differences between TD and the combined PRI group and no right-hemisphere M100 latency differences between ASD PRI-low and PRI-high.For M100, a main effect of hemisphere, s et al. , post hoTable p’s < 0.001). Added first, age accounted for a significant 16% variance in left M50 latency (p < 0.001) and a significant 11% variance in right M50 latency (p < 0.001). Added second, group added a marginally significant 2% variance in left M50 latency (p = 0.06) and a significant 4% variance in right M50 latency (p < 0.05). The group × age interaction was not significant in either hemisphere . Figure For each hemisphere, to examine how M50 and M100 latency differs as a function of group and age, hierarchical regressions were run in which age was entered first, diagnosis second, and their interaction last, with M50 or M100 latency as the dependent variable. For M50, in both hemispheres the full regression model  accounted for considerable variance in M50 latency . Added first, age accounted for a significant 28% variance in left M100 latency (p < 0.001) and a significant 25% variance in right M100 latency (p < 0.001). Added second, group added a non-significant 1% variance in left M100 latency (p > 0.05) and a marginally significant 3% variance in right M100 latency (p = 0.07). The group × age interaction was not significant in either hemisphere . Figure For M100, in both hemispheres, the full regression model  accounted for considerable variance in M100 latency , whereas grand average waveforms are shown for each ASD by year age group, grand averages were computed in 2-year steps for TD.Grand average left and right STG source waveforms are shown for ASD Figure  and TD  as a funExamination of the left (solid line) and right (dotted line) waveforms in the TD groups shows even in the youngest TD subjects a distinct left and right STG M100 . Examination of the ASD − LI and TD plots shows that the left STG M100 appears later in time , and only develops into a clearly distinct component in older subjects. For example, only in the 14- to 15-year-old TD group is the left M100 peak clearly distinct from M200. This is in contrast to the right M100, where even in the 6- to 7-year-old TD group, M100 is distinct from M200. With regard to hemisphere differences, the source waveforms suggest that only in the oldest ASD − LI and TD subjects is there, on average, similarity in latencies between the two hemispheres.Atypical auditory responses were observed in ASD. First, although STG M50 responses were observed in almost all subjects , however, have reported different group latency findings. Using a paired-click paradigm and examining P50 responses at electrode Cz, Orekhova et al.  observedpost hoc analyses examining only the right-hemisphere showed significant right-hemisphere M100 latency delays in ASD + LI and ASD − LI versus TD  than the TD and ASD − LI groups. Thus, lower cognitive ability rather than language impairment likely account for the present findings and thus the hypothesis that language impairment would best predict missing M100 responses was not supported. Finally, The above M50 and M100 abnormalities in ASD may reflect distinct auditory cortex abnormalities. Development of deep layers (lower layer III to layer VI) in auditory cortex occurs between 6 months and 5 years of age . Present findings also clearly indicate that developmental studies examining N100 activity at a single midline scalp site  are problematic as latency and amplitude measures at a single site in any individual could reflect activity from only a single hemisphere or, more likely, from some n et al.  and Sussn et al.  note than et al.  for a moIt is possible that present findings could be improved via co-registering each subject to their own MRI and then using anatomical constraints  to place dipoles, a strategy that will be examined in future studies. Although possible in older children, as it is often not possible to obtain structural MR data in infants and young children, for some studies it will still be necessary to apply techniques to align MEG data to template MRIs. In future studies examining younger children, the use of whole-brain infant and young child MEG systems will be preferred, with a smaller helmet size providing optimal signal-to-noise in younger children , MEG does not easily detect radial sources. As such, studies using EEG (or simultaneous EEG + MEG) are needed to examine radially oriented STG auditory sources (e.g., see Ponton et al., Finally, present findings as well as other studies indicate the need for longitudinal studies to more fully understand the development of auditory responses. For example, it has been reported that in some subjects, two M50 responses are observed. For example, using source modeling to examine left and right STG activity in children aged 7- to 16-years-old, Orekhova et al.  observedAlthough almost all TD and ASD subjects had a M50 response, M50 responses were delayed in ASD than TD bilaterally. Although M100 latencies were longer in the left- than right-hemisphere in TD, this delay was not so pronounced such that even young TD subjects had an identifiable left and right M100 by 6 years of age. Whereas there was a significant increase in the presence of M100 responses in the older than younger ASD − LI group, many individuals in the older ASD + LI group had a missing M100. Examining the TD data, present findings indicate that by 11 years, a right M100 should be observed in 100% of subjects and a left M100 in 80% of subjects. Thus, by 11 years old, if a long inter-trial interval is used, lack of a left and especially right M100 offers neurobiological insight into abnormal sensory processing that may underlie language or cognitive impairment in ASD.The Reviewer Elysa Jill Marco declares that despite having collaborated with the author Timothy P. L. Roberts on another project, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fnhum.2014.00417/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Background: Auditory processing and language impairments are prominent in children with autism spectrum disorder (ASD). The present study integrated diffusion MR measures of white-matter microstructure and magnetoencephalography (MEG) measures of cortical dynamics to investigate associations between brain structure and function within auditory and language systems in ASD. Based on previous findings, abnormal structure-function relationships in auditory and language systems in ASD were hypothesized.Methods: Evaluable neuroimaging data was obtained from 44 typically developing (TD) children (mean age 10.4 ± 2.4 years) and 95 children with ASD (mean age 10.2 ± 2.6 years). Diffusion MR tractography was used to delineate and quantitatively assess the auditory radiation and arcuate fasciculus segments of the auditory and language systems. MEG was used to measure (1) superior temporal gyrus auditory evoked M100 latency in response to pure-tone stimuli as an indicator of auditory system conduction velocity, and (2) auditory vowel-contrast mismatch field (MMF) latency as a passive probe of early linguistic processes.Results: Atypical development of white matter and cortical function, along with atypical lateralization, were present in ASD. In both auditory and language systems, white matter integrity and cortical electrophysiology were found to be coupled in typically developing children, with white matter microstructural features contributing significantly to electrophysiological response latencies. However, in ASD, we observed uncoupled structure-function relationships in both auditory and language systems. Regression analyses in ASD indicated that factors other than white-matter microstructure additionally contribute to the latency of neural evoked responses and ultimately behavior. Results also indicated that whereas delayed M100 is a marker for ASD severity, MMF delay is more associated with language impairment.Conclusion: Present findings suggest atypical development of primary auditory as well as auditory language systems in ASD. Findings demonstrate the need for additional multimodal studies to better characterize the different structural features  that contribute to brain activity, both in typical development and in ASD. Finally, the neural latency measures were found to be of clinical significance, with M100 associated with overall ASD severity, and with MMF latency associated with language performance. The etiology or, indeed, etiologies of autism spectrum disorder (ASD) is currently unknown. It is hypothesized that alterations to brain structure and function contribute to the clinical symptoms common to ASD. Given the high rate of occurrence of communication and language impairments in ASD, research has focused on the brain regions associated with basic auditory processes and more complex language skills, with prior imaging studies showing alterations in temporal lobe structure, connectivity and function  measures of white-matter microstructure with magnetoencephalography (MEG) measures of cortical neural dynamics. Diffusion MRI is sensitive to microstructural properties of white matter such as axon density and myelination and has been used in quantitative studies of the superior temporal gyrus and arcuate fasciculus in ASD  of the thalamus to the primary/secondary auditory cortex of the superior temporal gyrus (STG). The second white matter pathway examined was the arcuate fasciculus (AF), a tract from the STG to higher-order language areas. These circuits represent relatively early stages of connection relevant for auditory processing and then later stages relevant for language functioning. To this end, the first functional MEG measure examined was the 100 ms (M100) STG auditory response. Auditory evoked M100 latency has been shown to be due, in part, to thalamocortical conduction velocity along the auditory radiation  children, with these associations less evident in children with ASD .Children with a prior clinical ASD diagnosis were recruited from the Regional Autism Center of The Children's Hospital of Philadelphia (CHOP) and from local parent support groups. During the research visit, clinical and diagnostic testing by licensed child psychologists were performed to confirm ASD diagnosis and to ensure that typically developing children met inclusion criteria. ASD diagnosis was confirmed with the th percentile on the CELF-4. All subjects scored at or above the second percentile (SS > 70) on either the Perceptual Reasoning Index (PRI) or the Verbal Comprehension Index (VCI) of the Wechsler Intelligence Scale for Children-IV (WISC-IV). Detailed inclusion and exclusion criteria of the ASD and TD groups have been described previously  = 0.35, p = 0.70]. Of the children with ASD with diagnostic language scores, 35 were classified as ASD with language impairment  and 56 as ASD without language impairment . Although, the difference in age between ASD/+LI and ASD/-LI was significant , the age difference was small. Given the challenges of multimodal imaging, complete diffusion MR and MEG datasets were not available for all subjects. In particular, 28 participants had evaluable HARDI and DTI for measurement of the auditory radiation as well as evaluable M100 latency. Eighty-two participants had evaluable MMF latency and 78 participants had evaluable DTI for measurement of the arcuate fasciculus.The pool of participants with evaluable neuroimaging data for this multimodal study included 44 TD (mean age 10.4 ± 2.4 years) and 95 ASD (mean age 10.2 ± 2.6 years). MEG and DTI findings from a smaller subset of these participants have been previously reported . The details of the M100 and MMF tasks and data processing have been previously described . HARDI and conventional DTI were performed. Whole-brain HARDI acquisition included 64 gradient directions at a priori hypothesis to reduce statistical confounds associated with multiple comparisons in more comprehensive explorations . A single “effective” M100 latency for each participant's left and right STG was calculated with a linear mixed model to reduce the number of M100 latency analyses  = 9.1, p = 0.004; RD: F = 11.8, p = 0.001; FA: F = 6.63, p = 0.013]. The main effect of group was not significant for any DTI parameter. Significant age by group interactions for FA and RD , indicated group differences in maturation. In particular, collapsing across hemisphere, FA increased by 0.013 per year  in TD vs. 0.001 per year  in ASD, and RD decreased by 15.2 × 10−6 mm2/s/year  in TD vs. 3.04 × 10−6 mm2/s/year  in ASD.Each DTI parameter was linearly modeled with main effects of group, age, and hemisphere, along with each two-way interaction term. Auditory radiation MD and RD decreased with age and FA increased with age, indicating white matter maturation across both ASD and TD in the studied age range , RD , and AD . As depicted in Figure left hemisphere FA increased at a faster rate in the TD than ASD, driving the overall group by age difference. The slower maturation of FA in ASD was likely due to a slower rate of RD decrease in ASD vs. TD. No differences in rate of maturation were detected in the right hemisphere.Although, there was no significant group by hemisphere by age interaction, given overall group maturation rate differences and  FA . M100 did not predict language ability (CELF-4). A mean adjusted leverage plot was used to isolate and visualize the relationship between SRS and M100 latency  were modeled with M100 latency, age, and hemisphere as factors. M100 was a significant predictor of SRS [F1, 7 = 6.1, F = 4.7, p = 0.04, showed that increased RD was related to longer M100 latencies (slope = 15.7 × 104 ms * s/mm2). A similar trend was observed between MD and M100 latency, F = 3.4, p = 0.08. Analyzing groups separately showed this structure-function relationship was primarily driven by the TD group: RD was positively correlated with M100 in TD, F = 6.2, p = 0.04 , but not in ASD, F = 0.8, p = 0.4 (slope = 10.4 × 104 ms * s/mm2).For the multimodal analysis, to account for additional variance in the M100 latency, a linear model of each participant's effective M100 latency with average DTI parameters and group as effects was constructed. Age was not included given the above results indicating that both DTI and M100 measures show age dependence and may have interdependent maturational trajectories. A main effect of RD, p's < 0.005 with Tukey–Kramer Adjustment for multiple comparisons). MMF latency was modeled with group, stimulus type (vowel /a/ or /u/), and hemisphere as fixed effects and subject as a random effect. Additionally, PRI was considered as a potential confounding covariate.Given the reported relationship between MMF latency and language ability, group comparisons of CELF-4 and MMF latency included three groups: ASD with and without language impairment  as well as TD. As expected, mean CELF-4 Core Language Index (CLI) scores were significantly different for all pairwise group comparisons  = 4.2, p = 0.02 . MMF latency did not differ with respect to stimulus type or hemisphere and was not associated with PRI or age. Within the combined ASD group, MMF latency (latency collapsed across hemisphere) correlated with CELF-4 CLI, F = 8.4, p = 0.01  = 7.5, p = 0.01 in left; F = 6.1, p = 0.01 in right. No significant association between MMF latency and language ability (CELF-4 CLI) was observed in TD. In contrast to M100 latency, no significant association between MMF latency and SRS was observed in ASD or TD.There was a main effect of group on MMF latency, F = 4.0, p = 0.05 (slope = 0.0024/year) and TD groups, F = 21.8, p < 0.0001 (slope = 0.0052/year). MD decreased with age in the ASD, F = 22.4, p < 0.0001 (slope = −0.052 × 10−4 mm2/s/year) and TD groups F = 28.6, p < 0.0001 (slope = −0.076 × 10−4 mm2/s/year). Group by age interactions were not significant, indicating no detectable group difference in FA or MD maturation slopes. DTI parameters (with age as a covariate) were not predictive of CELF-4 CLI in either ASD or TD.To examine the role of white-matter microstructure on MMF latency as well as language ability, arcuate fasciculus DTI parameters were measured. Arcuate fasciculus FA increased with age in the ASD, F = 4.2, p = 0.04, indicated associations between FA and MMF latency. Explored in each group, FA predicted MMF in TD, F = 4.60, p = 0.04  or in the ASD/+LI (p = 0.4) or ASD/-LI (p = 0.4) subgroups. No associations with MMF latency were observed for the other DTI parameters .To explore contributions to MMF latency by arcuate fasciculus microstructure, linear mixed models with MMF latency as the dependent variable were constructed with DTI parameter, PRI, group (ASD and TD), age, stimulus type, and hemisphere as main effects and subject as the random effect. A main effect of FA, 4 Figure , and notThe present multimodal and multi-circuit study examined associations between brain structure and function in auditory and language areas. In both auditory and language systems, white-matter integrity, and cortical electrophysiology were found to be coupled in TD, with white-matter microstructural features contributing to M100 and MMF latency. However, in ASD, these structure–function relationships were less obvious or uncoupled. Results also suggested that the neural latency measures are of clinical significance, with a delayed M100 associated with increased ASD severity (as measured with SRS) and a delayed MMF delay associated with greater language impairment (CELF-4 CLI).Accurate and rapid encoding of auditory information is critical for receptive language. A prior study of auditory processing observed abnormal brainstem and cortical electrophysiology in children with language learning problems , supported the hypothesis that MMF latency is an index of language impairment rather than ASD severity or intellectual ability  as well as later-stage auditory processing (MMF) in ASD. As white-matter microstructure did not explain all the variance in M100 and MMF latency, other aspects of brain structure clearly contribute to age-related changes in M100 and MMF latency. Future multimodal studies examining a broader array of brain structure measures  are needed to more fully understand auditory encoding impairments in children with ASD. Finally, the neural latency measures were found to be of clinical significance, with M100 associated with SRS (but not CELF-4 CLI) indicating that slow auditory processing is an indicator of overall ASD severity, and with MMF latency associated with language performance.JB, TR, JE, SL, LB, and EK were involved in study conception and design. All authors were involved in acquisition of MRI, MEG, or clinical scores. JB, TR, JE, LB, EK, JD, and MK were involved in the interpretation or analysis of data. JB, JE, and TR drafted the manuscript. All authors approved the final manuscript.This work was supported by K01MH096091, R01DC008871-07, and the CHOP/UPenn IDDRC grant U54 HD086984.JB is a consultant for McGowan Associates. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AM and the handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.