The Changing Landscape of Pediatric Viral Enteropathogens in the Post-Rotavirus Vaccine Era.

BACKGROUND: Acute gastroenteritis (AGE) is a common reason for children to receive medical care. However, the viral etiology of AGE illness is not well described in the post-rotavirus vaccine era, particularly in the outpatient (OP) setting. METHODS: Between 2012 and 2015, children 15 days through 17 years old presenting to Vanderbilt Children's Hospital, Nashville, Tennessee, with AGE were enrolled prospectively from the inpatient, emergency department, and OP settings, and stool specimens were collected. Healthy controls (HCs) were enrolled and frequency matched for period, age group, race, and ethnicity. Stool specimens were tested by means of reverse-transcription real-time quantitative polymerase chain reaction for norovirus, sapovirus, and astrovirus RNA and by Rotaclone enzyme immunoassay for rotavirus antigen, followed by polymerase chain reaction verification of antigen detection. RESULTS: A total of 3705 AGE case patients and 1563 HCs were enrolled, among whom 2885 case patients (78%) and 1110 HCs (71%) provided stool specimens that were tested. All 4 viruses were more frequently detected in AGE case patients than in HCs (norovirus, 22% vs 8%, respectively; rotavirus, 10% vs 1%; sapovirus, 10% vs 5%; and astrovirus, 5% vs 2%; P < .001 for each virus). In the OP setting, rates of AGE due to norovirus were higher than rate for the other 3 viruses. Children <5 years old had higher OP AGE rates than older children for all viruses. CONCLUSIONS: Norovirus remains the most common virus detected in all settings, occurring nearly twice as frequently as the next most common pathogens, sapovirus and rotavirus. Combined, norovirus, sapovirus, rotavirus, and astrovirus were associated with almost half of all AGE visits and therefore are an important reason for children to receive medical care.

Impact of Culture-Independent Diagnostic Testing on Recovery of Enteric Bacterial Infections.

Background: Culture-independent diagnostic tests (CIDTs) are increasingly used to identify enteric pathogens. However, foodborne illness surveillance systems have relied upon culture confirmation to estimate disease burden and identify outbreaks through molecular subtyping. This study examined the impacts of CIDT and estimated costs for culture verification of Shigella, Salmonella, Shiga toxin-producing Escherichia coli (STEC), and Campylobacter at the Tennessee Department of Health Public Health Laboratory (PHL). Methods: This observational study included laboratory and epidemiological surveillance data collected between years 2013-2016 from patients with the reported enteric illness. We calculated pathogen recovery at PHL based on initial diagnostic test type reported at the clinical laboratory. Adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs) were estimated with modified Poisson regression. Estimates of cost were calculated for pathogen recovery from CIDT-positive specimens compared to recovery from culture-derived isolates. Results: During the study period, PHL received 5553 specimens from clinical laboratories from patients with the enteric illness. Pathogen recovery was 57% (984/1713) from referred CIDT-positive stool specimens and 95% (3662/3840) from culture-derived isolates (PR, 0.61 [95% CI, .56-.66]). Pathogen recovery from CIDT-positive specimens varied based on pathogen type: Salmonella (72%), Shigella (64%), STEC (57%), and Campylobacter (26%). Compared to stool culture-derived isolates, the cost to recover pathogens from 100 CIDT-positive specimens was higher for Shigella (US $6192), Salmonella (US $18373), and STEC (US $27783). Conclusions: Pathogen recovery was low from CIDT-positive specimens for enteric bacteria. This has important implications for the current enteric disease surveillance system, outbreak detection, and costs for public health programs.

Clinical Microbiology Laboratories' Adoption of Culture-Independent Diagnostic Tests Is a Threat to Foodborne-Disease Surveillance in the United States.

INTRODUCTIONIn November 2015, the Centers for Disease Control and Prevention (CDC) sent a letter to state and territorial epidemiologists, state and territorial public health laboratory directors, and state and territorial health officials. In this letter, culture-independent diagnostic tests (CIDTs) for detection of enteric pathogens were characterized as "a serious and current threat to public health surveillance, particularly for Shiga toxin-producing Escherichia coli (STEC) and Salmonella" The document says CDC and its public health partners are approaching this issue, in part, by "reviewing regulatory authority in public health agencies to require culture isolates or specimen submission if CIDTs are used." Large-scale foodborne outbreaks are a continuing threat to public health, and tracking these outbreaks is an important tool in shortening them and developing strategies to prevent them. It is clear that the use of CIDTs for enteric pathogen detection, including both antigen detection and multiplex nucleic acid amplification techniques, is becoming more widespread. Furthermore, some clinical microbiology laboratories will resist the mandate to require submission of culture isolates, since it will likely not improve patient outcomes but may add significant costs. Specimen submission would be less expensive and time-consuming for clinical laboratories; however, this approach would be burdensome for public health laboratories, since those laboratories would need to perform culture isolation prior to typing. Shari Shea and Kristy Kubota from the Association of Public Health Laboratories, along with state public health laboratory officials from Colorado, Missouri, Tennessee, and Utah, will explain the public health laboratories' perspective on why having access to isolates of enteric pathogens is essential for public health surveillance, detection, and tracking of outbreaks and offer potential workable solutions which will allow them to do this. Marc Couturier of ARUP Laboratories and Melissa Miller of the University of North Carolina will explain the advantages of CIDTs for enteric pathogens and discuss practical solutions for clinical microbiology laboratories to address these public health needs.

Multidrug-Resistant Salmonella Heidelberg Associated with Mechanically Separated Chicken at a Correctional Facility.

We describe multidrug-resistant (MDR) Salmonella Heidelberg infections associated with mechanically separated chicken (MSC) served at a county correctional facility. Twenty-three inmates met the case definition. All reported diarrhea, 19 (83%) reported fever, 16 (70%) reported vomiting, 4 (17%) had fever ≥103°F, and 3 (13%) were hospitalized. A case-control study found no single food item significantly associated with illness. Salmonella Heidelberg with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from nine stool specimens; two isolates displayed resistance to a total of five drug classes, including the third-generation cephalosporin, ceftriaxone. MDR Salmonella Heidelberg might have contributed to the severity of illness. Salmonella Heidelberg indistinguishable from the outbreak subtype was isolated from unopened MSC. The environmental health assessment identified cross-contamination through poor food-handling practices as a possible contributing factor. Proper hand-washing techniques and safe food-handling practices were reviewed with the kitchen supervisor.

Bacterial enteric infections detected by culture-independent diagnostic tests--FoodNet, United States, 2012-2014.

The increased availability and rapid adoption of culture-independent diagnostic tests (CIDTs) is moving clinical detection of bacterial enteric infections away from culture-based methods. These new tests do not yield isolates that are currently needed for further tests to distinguish among strains or subtypes of Salmonella, Campylobacter, Shiga toxin-producing Escherichia coli, and other organisms. Public health surveillance relies on this detailed characterization of isolates to monitor trends and rapidly detect outbreaks; consequently, the increased use of CIDTs makes prevention and control of these infections more difficult. During 2012-2013, the Foodborne Diseases Active Surveillance Network (FoodNet*) identified a total of 38,666 culture-confirmed cases and positive CIDT reports of Campylobacter, Salmonella, Shigella, Shiga toxin-producing E. coli, Vibrio, and Yersinia. Among the 5,614 positive CIDT reports, 2,595 (46%) were not confirmed by culture. In addition, a 2014 survey of clinical laboratories serving the FoodNet surveillance area indicated that use of CIDTs by the laboratories varied by pathogen; only CIDT methods were used most often for detection of Campylobacter (10%) and STEC (19%). Maintaining surveillance of bacterial enteric infections in this period of transition will require enhanced surveillance methods and strategies for obtaining bacterial isolates.

The frequency and seasonality of influenza and other respiratory viruses in Tennessee: two influenza seasons of surveillance data, 2010-2012.

BACKGROUND: In 2010, the Tennessee Department of Health, in collaboration with the Centers for Disease Control and Prevention (CDC), expanded influenza surveillance in Tennessee to include other respiratory viruses. OBJECTIVES: To determine the prevalence and seasonality of influenza and other respiratory viruses during the influenza seasons of 2010-2012. METHODS: Nasal and nasopharangeal swabs/washings from persons with influenza-like illness were collected across Tennessee. Influenza and other respiratory viruses were identified using a molecular-based respiratory virus panel. Influenza A positives were subtyped using real-time PCR according to the CDC protocol. Data were analyzed to describe frequency and seasonality of circulating strains. RESULTS: Of the 933 positive specimens, 60·3% were identified as influenza viruses, 19·8% rhinovirus/enterovirus, 8·6% respiratory syncytial virus (RSV), 5·8% metapneumovirus, 3·0% adenovirus, and 2·5% parainfluenza viruses. In the 2010-2011 season, influenza B was prominent during weeks 48-3, while influenza A(H1N1) was most frequently identified during weeks 4-10. Influenza A(H3N2) was present at lower levels during weeks 48-17. However, in the 2011-2012 season, overall numbers of influenza cases were reduced and influenza A (H3N2) was the most abundant influenza strain. The expanded surveillance for other respiratory viruses noted an increase in identified specimens from the first to the second season for adenovirus, metapneumovirus, RSV, and rhinovirus/enterovirus. CONCLUSIONS: This study provides data of the influenza strains in circulation in Tennessee. It also establishes a baseline and time of year to expect other respiratory viruses that will aid in detecting outbreaks of non-influenza respiratory viruses in Tennessee.

Effects and clinical significance of GII.4 Sydney norovirus, United States, 2012-2013.

During 2012, global detection of a new norovirus (NoV) strain, GII.4 Sydney, raised concerns about its potential effect in the United States. We analyzed data from NoV outbreaks in 5 states and emergency department visits for gastrointestinal illness in 1 state during the 2012-13 season and compared the data with those of previous seasons. During August 2012-April 2013, a total of 637 NoV outbreaks were reported compared with 536 and 432 in 2011-2012 and 2010-2011 during the same period. The proportion of outbreaks attributed to GII.4 Sydney increased from 8% in September 2012 to 82% in March 2013. The increase in emergency department visits for gastrointestinal illness during the 2012-13 season was similar to that of previous seasons. GII.4 Sydney has become the predominant US NoV outbreak strain during the 2012-13 season, but its emergence did not cause outbreak activity to substantially increase from that of previous seasons.

Outbreak of late-onset group B Streptococcus in a neonatal intensive care unit.

BACKGROUND: In September 2007, the Tennessee Department of Health was notified of a cluster of late-onset group B streptococcal (GBS) infections in a neonatal intensive care unit (NICU). Outbreaks of late-onset GBS are rare. METHODS: A case was defined as culture-confirmed invasive GBS infection in a neonate aged > or =7 days, identified in hospital A during August 23 to September 6, 2007. We reviewed medical records; examined NICU microbiology reports; and performed serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) on invasive isolates. Maternal GBS screening, prophylaxis, and infection control policies were reviewed and staff practices observed. RESULTS: Five cases of late-onset GBS were identified. None of the mothers of the infants received optimal GBS prophylaxis. Patient isolates were of 2 serotypes, 3 PFGE patterns, and 2 MLST patterns. Three isolates were indistinguishable on subtyping. These 3 cases were clustered in time. No common health care providers were identified. Infection control deviations in the NICU were observed. CONCLUSION: We identified a multiclonal cluster of 5 late-onset GBS cases. Multiple factors likely contributed to the outbreak, including nosocomial transmission of GBS. Further efforts to prevent late-onset GBS disease are necessary.

Methicillin-resistant Staphylococcus aureus and skin infections among personnel at a pediatric clinic.

Ambulatory care visits for methicillin-resistant Staphylococcus aureus (MRSA) infections are increasing dramatically. We investigated a pediatric clinic worker's death caused by MRSA. Among 45 clinic personnel, 16 reported recent skin infections, and 4% were colonized with MRSA. Among 262 patients, 3.4% were colonized with MRSA. Standard precautions were inconsistently applied when treating skin infections. Eight (11%) of 71 environmental swipes contained S aureus. Health care workers in outpatient settings are increasingly exposed to substantial numbers of persons with MRSA, and infection control practices in the ambulatory care setting deserve reemphasis.

Development and evaluation of a 4-target multiplex real-time polymerase chain reaction assay for the detection and characterization of Yersinia pestis.

A multiplexed, 4-target real-time polymerase chain reaction (PCR) assay for the detection and characterization of Yersinia pestis was designed and optimized for respiratory and environmental samples. The target sequences include the entF3 gene of the chromosome, pla (plasminogen activator) on the pPCP1 virulence plasmid, caf1 (F1 capsule antigen) on the pMT1 virulence plasmid, and a region located on the pCD1 plasmid. The sensitivity of this assay was determined to be less than 85 CFU per reaction for each specimen type analyzed. This assay was also determined to be 100% specific with strains of Y. pestis, 9 additional Yersinia species, and related enteric and respiratory organisms. The results show that this multiplex real-time PCR assay using TaqMan(R) (Roche Molecular Systems, Inc., Alameda, CA) chemistry is sensitive and specific, requires minimal sample input, and can yield results in approximately 4 h. This assay is the first 4-target multiplex real-time PCR assay for Y. pestis in which detection and virulence assessment of Y. pestis can occur in one reaction, from clinical and environmental samples.

Family outbreaks of invasive community-associated methicillin-resistant Staphylococcus aureus infection.

Outbreaks of invasive methicillin-resistant Staphylococcus aureus infection within families are unusual. We investigated 2 family clusters of invasive methicillin-resistant Staphylococcus aureus infection, including 1 in which a young mother died of fulminant pneumonia. Although surveillance via culture of family contacts of patients with invasive methicillin-resistant Staphylococcus aureus infection is not currently recommended, such clusters should stimulate reevaluation of preventive measures.

Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas.

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins.

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.