DIAGNOSING UNKNOWN CHILDHOOD INFECTIONS

Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital

BRIEF HISTORY

Since January 2007, Texas Children’s Hospital (TCH) and its Department of Pathology have effectively partnered with the Grant-A-Starr Foundation (GASF). The Foundation contributed more than $1,200,000 to TCH Pathology during the first generation of this project. This investment has made a huge difference, impacting hundreds of children already in Houston. By 2011, we had implemented 13 new molecular diagnostic tests for rapid detection of respiratory viruses (including influenza H1N1), bacteria causing intestinal infections, viruses causing meningitis, and improved detection of HIV in children. Since 2011, 4 additional new molecular diagnostic tests have been added including respiratory viruses like Adenovirus and Rhinovirus. Many children have been saved and treated effectively by having the Right Diagnosis at the Right Time. The rapid response to the influenza H1N1 pandemic in 2009 highlighted the ability of our enhanced diagnostic R&D operation (supported by Grant-A-Starr) to help control the rapid spread of pandemic “flu” and manage these serious infections.

Texas Children’s and its Department of Pathology created the new Texas Children’s Microbiome Center (TCMC) in February 2009. With the support of GASF, the TCMC became fully operational in October 2011 in our new home (10,000 sq ft in the Feigin Tower at TCH). Dr. Ralph D. Feigin provided the original inspiration for this project, and it is appropriate that our efforts are now housed in the Feigin Tower at TCH. The TCMC serves as the home of our GASF supported project – Diagnosing Unknown Childhood Infections or the DUCI Project.

CURRENT CAPABILITIES

The TCMC has grown exponentially in recent years, with expanding personnel, technology, and capabilities. With the support of the GASF, we have continued to incorporate cutting edge technologies into our development and delivery of new diagnostics. The TCMC now houses two high-throughput next-generation sequencers and is awaiting the arrival of the first automated extraction instrument (the Qiagen Qiacube), which will allow us to more rapidly and robustly extract microbial DNA from a variety of different specimens obtained from patients. With our established sequencing pipelines, we routinely characterize the microbiome from various body sites, sequence complete genomes of microbes of interest, and we’ve even begun to explore the mycobiome (the fungal community). The TCMC has also consistently increased our bioinformatics capabilities, and we continue to explore new ways to integrate vital patient information with our laboratory data.

Sequencing genomes of microbes that commonly cause significant infections in children is also performed in the TCMC. In partnership with our Infectious Diseases physicians here at the hospital, we have sequenced hundreds of individual microbial genomes including Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), antibiotic-resistant Pseudomonas aeruginosa, and Clostridium difficile. These sequencing efforts allow us to track infections that may come from a common source as well as probe for developing antibiotic resistance, which is a serious concern in today’s healthcare environment. We are also utilizing our standard pipelines to identify pathogens that may be present at very low levels, such as in early infections in tissue samples where very few bacterial organisms can be detected.

Our center has also acquired grants funding translational research in high impact areas of pediatric care, including recurrent C. difficile infection (support from the NIH) and GI disorders in autism (support from Autism Speaks). Both of these efforts are helping to advance our development of new diagnostic approaches to difficult to detect pathogens and abnormal microbial communities, with the potential to improve opportunities for timely and effective treatment strategies.

DUCI PROJECT PRIORITIES:

1. Airways and Lungs – Up to 50 percent of airway/lung infections are unexplained, and the clinical impact (number of children affected) of respiratory tract infections makes this topic possibly the most pressing challenge in pediatric infectious diseases today. The TCMC has undertaken several projects focusing on discovery of the pulmonary microbiome in children including: RSV infections, the development of bronchopulmonary dysplasia (BPD) in premature infants, the development of bronchiolitis obliterans in pediatric lung transplant patients, aspiration in children, and the impact of chronic bacterial infections on the lungs of patients with cystic fibrosis (CF). In addition, we have also sequenced genomes of antimicrobial resistant organisms identified in our hospital (ex. Pseudomonas aeruginosa that is highly resistant to multiple antibiotics).

2. Sepsis, CNS, and Tissue-based Infections- We are currently optimizing our approach to samples that contain a very small number of microbes (commonly referred to as “low biomass” samples). This is highly important in the rapid detection of sepsis (bloodstream infections), where this overwhelming systemic or “whole body” infection may stem from pneumonia or other causes of infections. We also continue to examine the bloodstream and airway- lung-bloodstream connections for causes of unexplained infections. This endeavor also extends to cerebrospinal fluid. While CSF is considered “sterile”, more and more data are proving that microbes are present in every area of the body from birth and even during prenatal development. CSF specimens are being tested in parallel with conventional microbiology testing to determine the presence and impact of bacterial pathogens. These approaches are also being applied to tissue specimens obtained through the body where infection or an unfavorable shift in the microbiome (dysbiosis) is suspected.

3. The GI Tract- The gut microbiome has been well characterized by many groups, including our own, in a variety of patient populations and diseases. The GI tract also represents the first opportunity to utilize microbiome transplantation as a treatment and potential cure for bacterial infections. Here at TCH, microbiome transplantation is being deployed as a treatment for Clostridium difficile infection, and we routinely examine the microbiome pre- and post-treatment as it related to treatment success. We are also using gut microbiome profiles in the confirmation of GI symptoms, including in children with limited abilities to communicate abdominal pain (ex. autism).

THE FUTURE

With projects spanning all clinically relevant specimen types, the DUCI initiative continues to stay ahead of the curve. Microbiome characterization has been utilized for several years as a diagnostic tool in the screening of transplant donors as well as in the assessment of the patient pre- and post-transplant. Microbiome characterization will also be deployed later this year as a companion diagnostic to routine microbiology testing, providing valuable data on the role of pathogens in the complete microbial community.

The expansion of sequencing technologies has allowed for greater throughput, faster turnaround times (increasing the impact in real-time patient testing), and more extensive studies that employ whole genome sequencing (allowing us to explore strain level identification and comparisons) and RNA sequencing. We are working closely with our Infectious Diseases colleagues, and we anticipate continuing to impact patient care through these capabilities.

PROJECT LEADERS

 

James Versalovic, M.D., Ph.D., serves as Director of the TCMC and DUCI Project Director.

Ruth Ann Luna, Ph.D., serves as DUCI Project Co-Director and manages day-to-day project activities including lab personnel management. The core DUCI Project activities will center on Discovery and Development of new pediatric diagnostics. Dr. Luna serves as Assistant Professor and Director of Medical Metagenomics.

Jim Dunn, Ph.D., serves as Director of Medical Microbiology and Virology. In addition, he now directs the Clinical Microbiology Fellowship program that launched in July 2017.

 

OTHER FACULTY

Tor Savidge, Ph.D., adds expertise in metabolomics and basic scientific discovery. Dr. Savidge serves as Associate Professor and Associate Director of TCMC with a focus on Discovery.

Sridevi Devaraj, Ph.D., is Director of the Clinical Chemistry laboratory at TCH as serves as Professor and Associate Director of the TCMC with a focus on Translation.

Jennifer Spinler, Ph.D., adds expertise in microbial genomics and serves as Instructor within the TCMC.

 

ADDITIONAL PERSONNEL

The project is supported in the laboratory by a research coordinator, two research technicians, 2 research assistants, and a therapeutic microbiology fellow.

 

SELECT PUBLICATIONS

Luna RA, Boyanton BL, Mehta S, Courtney EM, Webb CW, Revell PA, and Versalovic J. (2011) Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children’s hospital. J Clin Microbiol 49:851-7.

Ing J, Mason E, Kaplan S, Lamberth L, Revell P, Luna RA, and Hulten K. (2012) Characterization of non-typeable Streptococcus pneumoniae pediatric isolates from 1994-2010. J Clin Microbiol. 50:1326-30.

Chandramohan L, Revell PA. (2012) Prevalence and molecular characterization of extended-spectrum-β-lactamase-producing Enterobacteriaceae in a pediatric patient population. Antimicrob Agents Chemother Sep;56(9):4765-70.

Buchan BW, Olson WJ, Pezewski M, Marcon MJ, Novicki T, Uphoff TS, Chandramohan L, Revell P, Ledeboer NA. (2013) Clinical Evaluation of a Real-time PCR Assay for the Identification of Salmonella, Shigella, Campylobacter (C. jejuni and C. coli), and Shiga Toxin Producing E. coli in Stool Specimens. J Clin Microbiol, Sep 18.

 

Luna RA, Millecker LA, Webb CR, Mason SK, Whaley EM, Starke JR, Hiatt PW, Versalovic J. (2013) Molecular epidemiological surveillance of multidrug-resistant Pseudomonas aeruginosa in a pediatric population of patients with cystic fibrosis and determination of risk factors for infection with the Houston-1 strain. J Clin Microbiol. 51(4):1237-40.

Chavan R, Pannaraj P, Luna RA, Szabo S, Adesina A, Versalovic J, Krance RA, Kennedy-Nasser AA. (2013) Significant morbidity and mortality attributable to Rothia mucilaginosa infections in children with hematologic malignancies or following hematopoietic stem cell transplantation. Pediatric Hematology and Oncology;30(5):445-54.

Chumpitazi BP, Hollister EB, Oezguen N, Tsai CN, McMeans A, Luna RA, Savidge TC, Versalovic J, Shulman RJ. (2014) Low fermentable substrate diet in children with irritable bowel syndrome: Pilot efficacy and microbiome relationships. Gut Microbes. Mar-Apr;5(2):165-75.

Lohmann P, Luna RA, Hollister EB, Devaraj S, Mistretta T-A, Welty SE, Versalovic J. (2014) The airways microbiome of intubated premature infants: Characteristics of changes that predict the development of bronchopulmonary dysplasia. Pediatric Research. Sep;76(3):294-301.

Kellermayer R, Nagy-Szakal D, Harris RA, Luna RA, Pitashny M, Schady D, Mir SAV, Lopez ME, Gilger MA, Belmont J, Hollister EB, Versalovic J. (2015). Serial fecal transplantation alters mucosal gene expression in pediatric ulcerative colitis. American Journal of Gastroenterology. Apr;110(4):604-6.

 

Davidovics ZH, Carter BA, Luna RA, Hollister EB, Shulman RJ, Versalovic J. (2016) The fecal microbiome in pediatric patients with short bowel syndrome. J Parenter Enteral Nutr.Nov;40(8)1106-1113.

Chumpitazi BP, Cope JL, Hollister EB, Tsai CM, McMeans AR, Luna RA, Versalovic J, Shulman RJ. (2015) Randomised clinical trial: Gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with irritable bowel syndrome. Alimentary Pharmacology & Therapeutics. Aug;42(4):418-27.

 

Gargis AS, Kalman L, Bick DP, da Silva C, Dimmock DP, Funke BH, Gowrisankar S, Hegde MR, Kulkarni S, Mason CE, Nagarajan R, Voelkerding KV, Worthey EA, Aziz N, Barnes J, Bennett SF, Bisht H, Church DM, Dimitrova Z, Gargis SR, Hafez N, Hambuch T, Hyland FCL, Luna RA, MacCannell D, Mann T, McCluskey MR, McDaniel TK, Ganova-Raeva LM, Rehm HL, Reid J, Campo DS, Resnick RB, Ridge PG, Salit ML, Skums P, Wong LC, Zehnbauer BA, Zook JM, Lubin IM. (2015) Good laboratory practice for next-generation sequencing informatics pipelines. Nature Biotechnology. Jul;33(7)689-693.

 

Hollister EB, Riehle K, Luna RA, Wiedler EM, Rubio-Gonzalez M, Mistretta T-A, Raza S, Doddapaneni HV, Metcalf GA, Muzny DM, Gibbs RA,  Petrosino J, Shulman RJ, Versalovic J. (2015) Structure and function of the healthy pre-adolescent pediatric gut microbiome. Microbiome Aug 26;3:36.

Boyanton BL Jr, Freji BJ, Robinson-Dunn B, Makin J, Runge JK, Luna RA. (2016) Neonatal Pasteurella multocida subsp. septica meningitis traced to household cats: Molecular linkage using repetitive sequence-based PCR (Rep-PCR). J Clin Microbiol. Jan 54(1):230-2.

Chehoud C, Dryga A, Hwang Y, Nagy-Szakal D, Hollister EB, Luna RA, Versalovic J, Kellermayer R, Bushman FD. (2016) Transfer of viral communities between human individuals during fecal microbiota transplantation. MBio. Mar 29;7(2):e00322.

Shulman RJ, Hollister EB, Cain K, Czyzewski DI, Self MM, Weidler EM, Devaraj S, Luna RA, Versalovic J, Heitkemper M. Psyllium fiber reduces abdominal pain in children with irritable bowel syndrome in a randomized, double-blind trial. Clin Gastroenterol Hepatol. 2016 Apr 11. pii: S1542-3565(16)30021-0.

Huang RS, Johnson CL, Pritchard L, Hepler R, Ton TT, Dunn JJ. (2016) Performance of the Verigene® enteric pathogens test, Biofire FilmArray™ gastrointestinal panel and Luminex xTAG® gastrointestinal pathogen panel for detection of common enteric pathogens. Diagn Microbiol Infect Dis Dec;86(4):336-339.

Preidis GA, Luna RA, Hollister EB, Schady D, Finegold MJ, Versalovic J, Shulman RJ. The mucosal microbiota in a young child with severe non-Helicobacter-gastritis. Therapeutic Advances in Gastroenterology. Sep;9(5):749-51.

 

Luna RA, Oezguen N, Balderas M, Venkatachalam A, Runge JK, Versalovic J, Veenstra-VanderWeele J, Anderson, GM, Savidge T, Williams KC. (2017)Distinct microbiome-neuroimmune signatures correlate with functional abdominal pain in children with autism spectrum disorders. Cellular and Molecular Gastroenterology and Hepatology. Dec 11;3(2):218-230.

 

Flores AR, Luna RA, Runge JK, Shelburne SA, Baker CJ. Cluster of fatal group A streptococcal emm87 infection within a single family: molecular basis for invasion and transmission. J Infect Dis. Apr 5, epub ahead of print.

 

 

SELECT PRESENTATIONS
Microbiome Center Overview – DUCI