Kym Pham

The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.

Summary The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture

Objective Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. Design Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. Results Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. Conclusions Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine. Clinical Trial Registration ClinicalTrial.gov NCT01577511.

Frequent transmission of the Mycobacterium tuberculosis Beijing lineage and positive selection for the EsxW Beijing variant in Vietnam

To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.Genomic analysis of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients identifies the transmission dynamics of Mtb in Vietnam including frequent transmission of Beijing lineage and positive selection for EsxW Beijing variant.

AmpliVar: Mutation Detection in High‐Throughput Sequence from Amplicon‐Based Libraries

Conventional means of identifying variants in high‐throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key‐value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low‐abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon‐based high‐throughput sequencing data. The method can be extended to enable alignment‐free confirmation of variants seen in hybridization capture target‐enrichment data.

Dynamics of the upper airway microbiome in the pathogenesis of asthma-associated persistent wheeze in preschool children

Repeated cycles of infection-associated lower airway inflammation drives the pathogenesis of persistent wheezing disease in children. Tracking these events across a birth cohort during their first five years, we demonstrate that >80% of infectious events indeed involve viral pathogens, but are accompanied by a shift in the nasopharyngeal microbiome (NPM) towards dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change in NPM frequently precedes the appearance of viral pathogens and acute symptoms. In non-sensitized children these events are associated only with “transient wheeze” that resolves after age three. In contrast, in children developing early allergic sensitization, they are associated with ensuing development of persistent wheeze, which is the hallmark of the asthma phenotype. This suggests underlying pathogenic interactions between allergic sensitization and antibacterial mechanisms.

Evaluating the Genomic Yield of a Single Endobronchial Ultrasound-guided Transbronchial Needle Aspiration in Lung Cancer: Meeting the Challenge of Doing More With Less

Background Minimally invasive techniques, including endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA), yield small specimens that are adequate for cytologic diagnosis of lung cancer, but also need to provide material for molecular analysis to guide treatment. The number of EBUS‐TBNA passes needed for mutation testing remains unclear. We sought to assess the adequacy of a single pass for genomic profiling of actionable mutations. Methods In a prospective observational study, paired samples from the same lesion were obtained from patients undergoing EBUS‐TBNA for lung cancer diagnosis/staging. Following tumor cell confirmation by rapid on‐site evaluation, a “reference” sample comprising ≥ 3 passes was obtained and formalin‐fixed paraffin‐embedded. A “study” sample comprising a single pass was taken and snap‐frozen. The primary outcome was DNA yield and quality from a single pass. The secondary outcome was diagnostic accuracy of a single pass for detecting actionable mutations. Results In 40 patients, single‐pass specimens yielded a mean 3.98 &mgr;g of highly intact DNA, well above the minimum threshold for targeted sequencing, which was performed in adenocarcinoma cases (n = 24). In 23 cases, there was 100% agreement in mutation status between reference and study samples. In 1 case, the reference sample failed to generate a molecular diagnosis owing to insufficient tumor cells; however, the study specimen identified a KRAS mutation. Tumor cell percentage in mutation‐positive specimens was 1% to 70%, suggesting that single‐pass samples detect mutations even when tumor cell content is low. Conclusion Single EBUS‐TBNA passes yield DNA of high quantity and quality with high accuracy for molecular profiling, irrespective of tumor cell content. Micro‐Abstract The minimum quantity of material obtained by needle‐based techniques to perform genetic profiling of lung cancer remains unclear. We show that a single endobronchial ultrasound‐guided needle aspiration yields sufficient DNA to support genetic analysis with high diagnostic accuracy for clinically important mutations. This has significant implications for procedural performance in lung cancer cases where genetic alterations are highly suspected.

Genomic analysis of Mycobacterium tuberculosis reveals complex etiology of tuberculosis in Vietnam including frequent introduction and transmission of Beijing lineage and positive selection for EsxW Beijing variant

Tuberculosis (TB) is a leading cause of death from infectious disease and the global burden is now higher than at any point in history. Despite coordinated efforts to control TB transmission, the factors contributing to its successful spread remain poorly understood. Vietnam is identified as one of 30 high burden countries for TB and MDR-TB with an incidence of 137 TB cases per 100,000 individuals in 2015. Recent phylogenomic analyses of the causative agent Mycobacterium tuberculosis (Mtb) in other high-prevalence regions have provided insights into the complex processes underlying TB transmission. Here we examine the transmission dynamics of Mtb isolated from TB patients in Ho Chi Minh City (HCMC), Vietnam via whole genome analysis of 1,635 isolates and comparison with 3,085 isolates from other locations. The genomic data reveal an underlying burden of disease caused by endemic Mtb Lineage 1 associated with activation of long-term latent infection, on top of which is overlaid a three-fold higher burden associated with introduction of exotic Lineage 2 and 4 Mtb strains. We identify frequent transfer of Beijing lineage Mtb into the country, which are associated with higher levels of transmission in this host population than endemic Lineage 1 Mtb. We identify a mutation in the secreted protein EsxW in Beijing strains that also appears to be under positive selection in other Mtb lineages, which could potentially contribute to the enhanced transmission of the Beijing lineage in Vietnamese and other host populations.

Cohort-wide characterization of the nasopharyngeal microbiome across year 1 in "high risk" infants by PCR and 16S rRNA gene deep sequencing: elucidation of viral-bacterial-host interactions driving early atopic asthma pathogenesis

Banerji, A; Busse, P; Shennak, M; Lumry, W; Davis-Lorton, M; Wedner, J; Jacobs, J; Baker, J; Bernstein, J; Lockey, R; Li, H; Craig, T; Cicardi, M; Riedl, M; Al-Ghazawi, A; Soo, C; Iarrobino, R; Sexton, D; TenHoor, C; Kenniston, J; Faucette, R; Biedenkapp, J; Chyung, Y; Adelman, B Masschusetts General Hospital, Boston, United States; Mount Sinai School of Medicine, New York, United States; Triumpharma, Amman, Jordan; AARA Research Center, Dallas, United States; Winthrop University Hospital, Mineola, United States; Washington University School of Medicine, St. Louis, United States; Allergy and Asthma Clinical Research, Walnut Creek, United States; Baker Allergy Asthma and Dermatology, Lake Oswego, United States; Univeristy of Cincinnati College of Medicine, Cincinnati, United States; University of South Florida, Tampa, United States; Institute for Asthma and Allergy, Chevy Chase, United States; Penn State Hershey Medical Center, Hersey, United States; University of Milan-Ospedale Luigi Sacco, Milan, Italy; University of California, San Diego, La Jolla, United States; Dyax Corp., Burlington, United States