Concordance between predicted HLA type using next generation sequencing data generated for non-HLA purposes and clinical HLA type.

We explored the feasibility of obtaining accurate HLA type using pre-existing NGS data not generated for HLA purposes. 83 exomes and 500 targeted NGS pharmacogenomic panels were analyzed using Omixon HLA Explore, OptiType, and/or HLA-Genotyper software. Results were compared against clinical HLA genotyping. 765 (94.2%) Omixon and 769 (94.7%) HLA-Genotyper of 812 germline allele calls across class I/II loci and 402 (99.5%) of 404 OptiType class I calls were concordant to the second field (i.e. HLA-A*02:01). An additional 19 (2.3%) Omixon, 39 (4.8%) HLA-Genotyper, and 2 (0.5%) OptiType allele calls were first field concordant (i.e. HLA-A*02). Using Omixon, four alleles (0.4%) were discordant and 24 (3.0%) failed to call, while 4 alleles (0.4%) were discordant using HLA-Genotyper. Tumor exomes were also evaluated and were 85.4%, 91.6%, and 100% concordant (Omixon and HLA-Genotyper with 96 alleles tested, and Optitype with 48 class I alleles, respectively). The 15 exomes and 500 pharmacogenomic panels were 100% concordant for each pharmacogenomic allele tested. This work has broad implications spanning future clinical care (pharmacogenomics, tumor response to immunotherapy, autoimmunity, etc.) and research applications.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for interoperability, as performed by users with variable experience.

BACKGROUND: A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility. METHODS: Technologists from six laboratories typed the same 16 samples at HLA-A, B, C, DRB1, and DQB1. The protocol includes long range PCR, library preparation, and paired-end 250bp sequencing. Two indexing strategies were employed: locus-specific indexing whereby each locus was tagged uniquely and sample-specific indexing whereby all 5 loci for a sample were pooled prior to library preparation. Sequence analysis was performed with two software packages, Target HLA (Omixon) and NGSengine (GenDx). RESULTS: The average number of sequence reads per library was 387,813; however, analysis was limited to 40,000 reads for locus-indexed libraries and 200,000 reads for sample-indexed libraries resulting in an average depth of coverage of 1444 reads per locus. Sufficient reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy was >97% in pooled amplicons and >99% in individual amplicons by both software analysis. Inter-laboratory reproducibility was 99.7% and only cause of discordance was cross-contamination of a single amplicon. CONCLUSIONS: This NGS HLA-typing protocol is simple, reproducible, scalable, highly accurate and amenable to clinical testing.

Role of the Gut Microbiota of Children in Diarrhea Due to the Protozoan Parasite Entamoeba histolytica.

BACKGROUND: An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia. METHODS: We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh. RESULTS: Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea. CONCLUSIONS: E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota.

Evidence for diminished multisensory integration in autism spectrum disorders.

Individuals with autism spectrum disorders (ASD) exhibit alterations in sensory processing, including changes in the integration of information across the different sensory modalities. In the current study, we used the sound-induced flash illusion to assess multisensory integration in children with ASD and typically-developing (TD) controls. Thirty-one children with ASD and 31 age and IQ matched TD children (average age = 12 years) were presented with simple visual (i.e., flash) and auditory (i.e., beep) stimuli of varying number. In illusory conditions, a single flash was presented with 2-4 beeps. In TD children, these conditions generally result in the perception of multiple flashes, implying a perceptual fusion across vision and audition. In the present study, children with ASD were significantly less likely to perceive the illusion relative to TD controls, suggesting that multisensory integration and cross-modal binding may be weaker in some children with ASD. These results are discussed in the context of previous findings for multisensory integration in ASD and future directions for research.

Multisensory temporal integration in autism spectrum disorders.

The new DSM-5 diagnostic criteria for autism spectrum disorders (ASDs) include sensory disturbances in addition to the well-established language, communication, and social deficits. One sensory disturbance seen in ASD is an impaired ability to integrate multisensory information into a unified percept. This may arise from an underlying impairment in which individuals with ASD have difficulty perceiving the temporal relationship between cross-modal inputs, an important cue for multisensory integration. Such impairments in multisensory processing may cascade into higher-level deficits, impairing day-to-day functioning on tasks, such as speech perception. To investigate multisensory temporal processing deficits in ASD and their links to speech processing, the current study mapped performance on a number of multisensory temporal tasks (with both simple and complex stimuli) onto the ability of individuals with ASD to perceptually bind audiovisual speech signals. High-functioning children with ASD were compared with a group of typically developing children. Performance on the multisensory temporal tasks varied with stimulus complexity for both groups; less precise temporal processing was observed with increasing stimulus complexity. Notably, individuals with ASD showed a speech-specific deficit in multisensory temporal processing. Most importantly, the strength of perceptual binding of audiovisual speech observed in individuals with ASD was strongly related to their low-level multisensory temporal processing abilities. Collectively, the results represent the first to illustrate links between multisensory temporal function and speech processing in ASD, strongly suggesting that deficits in low-level sensory processing may cascade into higher-order domains, such as language and communication.

Brief report: Arrested development of audiovisual speech perception in autism spectrum disorders.

Atypical communicative abilities are a core marker of Autism Spectrum Disorders (ASD). A number of studies have shown that, in addition to auditory comprehension differences, individuals with autism frequently show atypical responses to audiovisual speech, suggesting a multisensory contribution to these communicative differences from their typically developing peers. To shed light on possible differences in the maturation of audiovisual speech integration, we tested younger (ages 6-12) and older (ages 13-18) children with and without ASD on a task indexing such multisensory integration. To do this, we used the McGurk effect, in which the pairing of incongruent auditory and visual speech tokens typically results in the perception of a fused percept distinct from the auditory and visual signals, indicative of active integration of the two channels conveying speech information. Whereas little difference was seen in audiovisual speech processing (i.e., reports of McGurk fusion) between the younger ASD and TD groups, there was a significant difference at the older ages. While TD controls exhibited an increased rate of fusion (i.e., integration) with age, children with ASD failed to show this increase. These data suggest arrested development of audiovisual speech integration in ASD. The results are discussed in light of the extant literature and necessary next steps in research.

The effect of antithymocyte globulin on anti-human leukocyte antigen antibody detection assays.

BACKGROUND: We evaluated the effect of antithymocyte globulin (ATG) on anti-human leukocyte antigen (HLA) antibody assays. METHODS: We tested sera from six in vivo ATG-treated kidney transplant patients after measuring serum concentrations, as well as six nonsensitized sera with ATG added in vitro. T- and B-cell complement-dependent cytotoxicity (CDC), flow cytometric (FXM), and solid-phase HLA class I and II assays based on antigen-coated microspheres and enzyme-linked immunosorbent assay (ELISA) were studied. Sera were then retested after treatment to remove ATG. RESULTS: We found that ATG affects test results differently depending on whether sera is obtained from in vivo treated patients or added in vitro. In vitro treated sera produced ATG concentration-dependent positive results for T/B CDC, FXM, and flow bead testing for HLA I/II, while the ELISA-based assay was unaffected. In vivo treated sera from ATG-treated patients produced positive test results for T CDC and T/B FXM, while the B-cell CDC crossmatch remained negative. Solid phase assays were minimally affected using in vivo treated sera. After ATG extraction, all tests became negative. CONCLUSION: We conclude that ATG produces positive results in anti-HLA antibody testing, and treatment to remove ATG abolishes this effect. This treatment allows ATG-treated patients to be monitored for anti-HLA antibodies.