Identification of Enteropathogens by Multiplex PCR among Rural and Urban Guatemalan Children with Acute Diarrhea.

Multiplex polymerase chain reaction (PCR) platforms have enhanced understanding of intestinal pathogens in low- and middle-income countries (LMICs). However, few such studies have been performed in Latin America, where poverty, poor sanitation, and undernutrition persist. Multiplex PCR (BioFire, Salt Lake City, UT) was used to identify viral, bacterial, and parasitic pathogens in stool collected on day 1 and 31 from children aged 6 to 35 months with acute, non-bloody diarrhea in two locations (rural and urban) in Guatemala. We analyzed correlation between pathogens and clinical, demographic, and socioeconomic variables; described patterns of pathogen acquisition, persistence, and clearance over the 30-day period; and calculated population attributable fractions (PAFs) for diarrheal causation for individual pathogens. We analyzed 316 subjects (144 urban; 172 rural) enrolled between March 2015 and January 2016. Rural subjects had significantly more malnutrition, animal exposure, and unimproved water/sanitation infrastructure. The majority of subjects had multiple pathogens/sample (4.8 rural and 2.7 urban). Few meaningful correlates were identified between individual pathogens and clinical, demographic, or environmental variables. Escherichia coli pathotypes, Shigella, Campylobacter, and Giardia had high rates of persistence between initial and 30-day follow-up. Statistically significant adjusted PAFs were identified for Campylobacter (14.9%, 95% CI: 3.2-23.1), norovirus (10.2%, 95% CI: 0.4-17.1), sapovirus (7.6%, 95% CI: 2.3-10.9), and adenovirus 40/41 (5.6%, 95% CI: 0.3-8.7). These observations further characterize the diversity and complexity of enteric pathogens in children in LMICs. Patterns of chronic symptomatic and asymptomatic infection among Latin American children are similar to those observed in other LMIC regions. Findings have direct implications for practitioners treating individuals with acute infectious diarrhea and should inform regional public health strategies.

Validation of a novel molecular diagnostic panel for pediatric musculoskeletal infections: Integration of the Cepheid Xpert MRSA/SA SSTI and laboratory-developed real-time PCR assays for clindamycin resistance genes and Kingella kingae detection.

BACKGROUND: Pathogen detection in pediatric patients with musculoskeletal infections relies on conventional bacterial culture, which is slow and can delay antimicrobial optimization. The ability to rapidly identify causative agents and antimicrobial resistance genes in these infections may improve clinical care. METHODS: Convenience specimens from bone and joint samples submitted for culture to Children's Hospital Colorado (CHCO) from June 2012 to October 2016 were evaluated using a "Musculoskeletal Diagnostic Panel" (MDP) consisting of the Xpert MRSA/SA SSTI real-time PCR (qPCR, Cepheid) and laboratory-developed qPCRs for Kingella kingae detection and erm genes A, B, and C which confer clindamycin resistance. Results from the MDP were compared to culture and antimicrobial susceptibility testing (AST) results. RESULTS: A total of 184 source specimens from 125 patients were tested. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA/SA SSTI compared to culture and AST results were 85%, 98%, 93%, and 95% respectively for MSSA and 82%, 100%, 100%, and 99% for MRSA. Compared to phenotypic clindamycin resistance in S. aureus isolates, the erm A, B, and C gene PCRs collectively demonstrated a sensitivity, specificity, PPV, and NPV of 80%, 96%, 67%, and 98%. In comparison to clinical truth, Kingella PCR had a sensitivity, specificity, PPV, and NPV of 100%, 99.5%, 100%, and 100%. CONCLUSIONS: This novel MDP offers a rapid, sensitive, and specific option for pathogen detection in pediatric patients with musculoskeletal infections.

Automated Real-Time Collection of Pathogen-Specific Diagnostic Data: Syndromic Infectious Disease Epidemiology.

BACKGROUND: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

Cytomegalovirus Retinitis in Pediatric Stem Cell Transplants: Report of a Recent Cluster and the Development of a Screening Protocol.

PURPOSE: The incidence of cytomegalovirus (CMV) retinitis in the pediatric allogeneic hematopoietic stem cell transplant (HSCT) population is unknown. We report a cluster of 5 pediatric patients with CMV retinitis diagnosed in a 12-month period and compare this to the rate of CMV viremia and retinitis in the 4 years prior. Presented is the ophthalmic screening protocol developed in response to this experience. DESIGN: Retrospective cross-sectional study. METHODS: A retrospective chart review was performed on patients at Children's Hospital of Colorado (CHCO) who received allogeneic HSCT between January 2010 and December 2014. Fisher exact test was used to compare the proportion of CMV viremia and CMV retinitis in patients transplanted between January 2010 and December 2013 with those transplanted in 2014. RESULTS: A total of 101 patients underwent allogeneic HSCT from January 2010 to December 2013; 32 (32%) tested positive for CMV viremia. No cases of CMV retinitis were identified. From January 2014 to December 2014, 28 patients underwent allogeneic HSCT; 13 patients (46%) had CMV viremia, not a statistically significant increase (P = .18). There were 5 cases of CMV retinitis diagnosed in those transplanted in 2014, a statistically significant increase compared with those transplanted in 2010-2013 (P = .0004). A multidisciplinary team was formed to review the literature and an ophthalmic screening protocol was developed. CONCLUSION: Our recent cluster of CMV retinitis in pediatric allogeneic HSCT patients may suggest a rise in incidence of CMV retinitis. We propose an ophthalmic screening protocol to diagnose retinitis in pediatric HSCT patients in the early, often asymptomatic stage.

Viral gastroenteritis in children in Colorado 2006-2009.

Acute gastroenteritis accounts for a significant burden of medically attended illness in children under the age of five. For this study, four multiplex reverse transcription PCR assays were used to determine the incidence of adenovirus, astrovirus, coronavirus, norovirus GI and GII, rotavirus, and sapovirus in stool samples submitted for viral electron microscopy (EM) to the Children's Hospital Colorado. Of 1105 stool samples available, viral RNA/DNA was detected in 247 (26.2%) of 941 pediatric samples (median age = 2.97 years, 54% male) with 28 (3.0%) positive for more than one virus. Adenovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, and sapovirus were detected in 95 (10.0%), 33 (3.5%), 8 (0.9%), 90 (9.6%), 49 (5.2%), and 2 (0.2%) of the pediatric samples, respectively. No coronaviruses were identified. Sequencing of norovirus positive samples indicated an outbreak of norovirus strain GII.4 in 2006 with evidence of numerous circulating strains. Multiple samples from the same immunocompromised patients demonstrated symptomatic shedding of norovirus for up to 32 weeks and astrovirus for 12 weeks. RT-PCR detected 99 of 111 (89%) adenovirus-positive samples versus 12 (11%) by EM, and 186 of 192 (97%) sapovirus/astrovirus/norovirus-positive samples versus 21 (11%) by EM. Noroviruses and adenoviruses are common causes of gastroenteritis in children. Immunocompromised patients can be infected with multiple viruses and shed viruses in their stools for prolonged periods. This data support the superiority of RT-PCR compared to EM for diagnosis of viral gastroenteritis.

Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination.

This study compared the complete genome sequences of 16 NL63 strain human coronaviruses (hCoVs) from respiratory specimens of paediatric patients with respiratory disease in Colorado, USA, and characterized the epidemiology and clinical characteristics associated with circulating NL63 viruses over a 3-year period. From 1 January 2009 to 31 December 2011, 92 of 9380 respiratory specimens were found to be positive for NL63 RNA by PCR, an overall prevalence of 1 %. NL63 viruses were circulating during all 3 years, but there was considerable yearly variation in prevalence and the month of peak incidence. Phylogenetic analysis comparing the genome sequences of the 16 Colorado NL63 viruses with those of the prototypical hCoV-NL63 and three other NL63 viruses from the Netherlands demonstrated that there were three genotypes (A, B and C) circulating in Colorado from 2005 to 2010, and evidence of recombination between virus strains was found. Genotypes B and C co-circulated in Colorado in 2005, 2009 and 2010, but genotype A circulated only in 2005 when it was the predominant NL63 strain. Genotype C represents a new lineage that has not been described previously. The greatest variability in the NL63 virus genomes was found in the N-terminal domain (NTD) of the spike gene (nt 1-600, aa 1-200). Ten different amino acid sequences were found in the NTD of the spike protein among these NL63 strains and the 75 partial published sequences of NTDs from strains found at different times throughout the world.

WU and KI polyomavirus infections in pediatric hematology/oncology patients with acute respiratory tract illness.

BACKGROUND: WU and KI polyomaviruses (PyV) were discovered in 2007 in respiratory tract samples in adults and children. Other polyomaviruses (BKPyV and JCPyV) have been associated with illness in immunocompromised patients, and some studies suggest a higher prevalence of WUPyV and KIPyV in this population. OBJECTIVE: To determine whether a higher prevalence or viral load for WUPyV and KIPyV exists in immunocompromised children compared with immunocompetent children. STUDY DESIGN: We measured the prevalence and viral load of WU and KI PyV by quantitative real-time PCR of viral DNA in respiratory tract specimens from pediatric hematology/oncology patients and immunocompetent controls with acute respiratory illnesses. RESULTS: The prevalence of WUPyV in the immunocompromised population was 5/161 (3%) versus 14/295 (5%) in the control population (P=0.5), and 9/161 (5.6%) versus 7/295 (2.3%) respectively for KIPyV (P=0.13). The mean viral load (in copies per cell or mL of sample) for KIPyV, was higher in the immunocompromised group compared to the control group (P=0.019), but was not statistically different for WUPyV. A higher prevalence was seen in the hematopoietic stem cell transplant recipients compared with other immunocompromised patients (6/26 versus 3/43, P=0.054). Viral persistence was demonstrated only in 1/25 (4%) of sequential samples for KIPyV, and no persistence was seen for WUPyV. CONCLUSIONS: A higher prevalence of WUPyV or KIPyV in the immunocompromised population compared with the immunocompetent group was not demonstrated. Higher viral loads for KIPyV in the immunocompromised group may suggest an increased pathogenic potential in this population.

Genotype prevalence and risk factors for severe clinical adenovirus infection, United States 2004-2006.

BACKGROUND: Recently, epidemiological and clinical data have revealed important changes with regard to clinical adenovirus infection, including alterations in antigenic presentation, geographical distribution, and virulence of the virus. METHODS: In an effort to better understand the epidemiology of clinical adenovirus infection in the United States, we adopted a new molecular adenovirus typing technique to study clinical adenovirus isolates collected from 22 medical facilities over a 25-month period during 2004-2006. A hexon gene sequence typing method was used to characterize 2237 clinical adenovirus-positive specimens, comparing their sequences with those of the 51 currently recognized prototype human adenovirus strains. In a blinded comparison, this method performed well and was much faster than the classic serologic typing method. RESULTS: Among civilians, the most prevalent adenovirus types were types 3 (prevalence, 34.6%), 2 (24.3%), 1 (17.7%), and 5 (5.3%). Among military trainees, the most prevalent types were types 4 (prevalence, 92.8%), 3 (2.6%), and 21 (2.4%). CONCLUSIONS: For both populations, we observed a statistically significant increasing trend of adenovirus type 21 detection over time. Among adenovirus isolates recovered from specimens from civilians, 50% were associated with hospitalization, 19.6% with a chronic disease condition, 11% with a bone marrow or solid organ transplantation, 7.4% with intensive care unit stay, and 4.2% with a cancer diagnosis. Multivariable risk factor modeling for adenovirus disease severity found that age <7 years (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4-7.4), chronic disease (OR, 3.6; 95% CI, 2.6-5.1), recent transplantation (OR, 2.7; 95% CI, 1.3-5.2), and adenovirus type 5 (OR, 2.7; 95% CI, 1.5-4.7) or type 21 infection (OR, 7.6; 95% CI, 2.6-22.3) increased the risk of severe disease.