Clinical Impact of Plasma Metagenomic Next-generation Sequencing in a Large Pediatric Cohort.

BACKGROUND: Plasma metagenomic next-generation sequencing (mNGS) has the potential to detect thousands of different organisms with a single test. There are limited data on the real-world impact of mNGS and even less guidance on the types of patients and clinical scenarios in which mNGS testing is beneficial. METHODS: A retrospective review of patients who had mNGS testing as part of routine clinical care at Texas Children's Hospital from June 2018-August 2019 was performed. Medical records were reviewed for pertinent data. An expert panel of infectious disease physicians adjudicated each unique organism identified by mNGS for clinical impact. RESULTS: There were 169 patients with at least one mNGS test. mNGS identified a definitive, probable or possible infection in 49.7% of patients. mNGS led to no clinical impact in 139 patients (82.2%), a positive impact in 21 patients (12.4%), and a negative impact in 9 patients (5.3%). mNGS identified a plausible cause for infection more often in immunocompromised patients than in immunocompetent patients (55.8% vs. 30.0%, P = 0.006). Positive clinical impact was highest in patients with multiple indications for testing (37.5%, P = 0.006) with deep-seated infections, overall, being most often associated with a positive impact. CONCLUSION: mNGS testing has a limited real-world clinical impact when ordered indiscriminately. Immunocompromised patients with well-defined deep-seated infections are likely to benefit most from testing. Further studies are needed to evaluate the full spectrum of clinical scenarios for which mNGS testing is impactful.

Risk factors for microbiologic failure in children with Enterobacter species bacteremia.

BACKGROUND: Enterobacter species are an important cause of healthcare-associated bloodstream infections (BSI) in children. Up to 19% of adult patients with Enterobacter BSI have recurrence of infection resistant to third-generation cephalosporins (3GCs) while on therapy with a 3GC. Data are lacking regarding the incidence of and risk factors for recurrence of infection in children with Enterobacter BSI. METHODS: We conducted a retrospective case-control study of patients aged ≤21 years old admitted to Texas Children's Hospital from January 2012 through December 2018 with Enterobacter BSI. The primary outcome was microbiologic failure from 72 hours to 30 days after the initial BSI (cases). The secondary outcome was isolation of a 3GC non-susceptible Enterobacter sp. from a patient with an initial 3GC-susceptible isolate. RESULTS: Twelve patients (6.7%) had microbiologic failure compared to 167 controls without microbiologic failure. Of the 138 patients (77.1%) with an Enterobacter sp. isolate that was initially susceptible to 3GCs, 3 (2.2%) developed a subsequent infection with a non-susceptible isolate. Predictors of microbiologic failure were having an alternative primary site of infection besides bacteremia without a focus or an urinary tract infection (OR, 9.64; 95% CI, 1.77-52.31; P < 0.01) and inadequate source control (OR, 22.16; 95% CI, 5.26-93.36; P < 0.001). CONCLUSIONS: Source of infection and adequacy of source control are important considerations in preventing microbiologic failure. In-vitro susceptibilities can be used to select an antibiotic regimen for the treatment of Enterobacter BSI in children.

Healthcare-associated Pediatric Cutaneous Mucormycosis at Texas Children's Hospital, 2012-2019.

Cutaneous mucormycosis in children is an opportunistic fungal infection associated with significant morbidity and mortality. We describe characteristics of 12 patients with healthcare-associated cutaneous mucormycosis at Texas Children's Hospital and results of an outbreak investigation. A definitive source was not identified. Skin lesions near medical device securement sites should raise concern for mucormycosis in patients with underlying medical conditions.

A Surge in Pediatric Coronavirus Disease 2019 Cases: The Experience of Texas Children's Hospital From March to June 2020.

BACKGROUND: An understanding of the clinical characteristics of children with coronavirus disease 2019 in diverse communities is needed to optimize the response of healthcare providers during this pandemic. METHODS: We performed a retrospective review of all children presenting to the Texas Children's Hospital system with testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from March 10, 2020, through June 28, 2020. Demographics were recorded for all patients undergoing testing and clinical characteristics and outcomes were recorded for children with positive tests. RESULTS: Of 16 554 unique patients ≤ 21 years of age who were tested for SARS-CoV-2, 1215 (7.3%) patients tested positive. Infants under 1 year of age and patients aged 18-21 years had the highest percent of positive tests at 9.9% (230/2329) and 10.7% (79/739), respectively. Hispanic children accounted for 66% (802/1215) of positive tests, though they only represented 42.1% (6972/16 554) of all children tested for SARS-CoV-2. Of the 1215 children with a positive test, 55.7% had fever, 40.9% had cough, 39.8% had congestion or rhinorrhea, 21.9% had gastrointestinal complaints, and 15.9% were asymptomatic. Only 97 (8%) patients were hospitalized (of which 68% were Hispanic). Most of the hospitalized patients had underlying medical conditions (62/97, 63.9%), including obesity. Thirty-one hospitalized patients (31/97, 32%) required respiratory support and 9 patients (9/97, 9.3%) received SARS-CoV-2 antiviral therapy. Two patients died. CONCLUSIONS: A relatively high percentage of Hispanic children tested positive for SARS-CoV-2 and were hospitalized. Most of the children with detection of SARS-CoV-2 had uncomplicated illness courses; some children were critically ill; and 2 patients died.

Update in Pediatric Diagnostic Microbiology.

Infants and young children are uniquely susceptible to primary viral and bacterial infections, predisposing them to responses of greater frequency and severity than in adults. Etiologies and manifestations of infections in pediatric patients are often different than those in adults. It can be challenging for clinical laboratories to implement appropriate microbiologic methods for rapid and accurate diagnoses in this population. Laboratorians should be cognizant of the distinctive features of children to provide comprehensive pediatric clinical microbiology services. This article discusses laboratory aspects of several clinically significant pediatric pathogens that cause severe harm to patients and impact public health responses.

Comparison of Upper Respiratory Viral Load Distributions in Asymptomatic and Symptomatic Children Diagnosed with SARS-CoV-2 Infection in Pediatric Hospital Testing Programs.

The distribution of upper respiratory viral loads (VL) in asymptomatic children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We assessed PCR cycle threshold (Ct) values and estimated VL in infected asymptomatic children diagnosed in nine pediatric hospital testing programs. Records for asymptomatic and symptomatic patients with positive clinical SARS-CoV-2 tests were reviewed. Ct values were (i) adjusted by centering each value around the institutional median Ct value from symptomatic children tested with that assay and (ii) converted to estimated VL (numbers of copies per milliliter) using internal or manufacturer data. Adjusted Ct values and estimated VL for asymptomatic versus symptomatic children (118 asymptomatic versus 197 symptomatic children aged 0 to 4 years, 79 asymptomatic versus 97 symptomatic children aged 5 to 9 years, 69 asymptomatic versus 75 symptomatic children aged 10 to 13 years, 73 asymptomatic versus 109 symptomatic children aged 14 to 17 years) were compared. The median adjusted Ct value for asymptomatic children was 10.3 cycles higher than for symptomatic children (P < 0.0001), and VL were 3 to 4 logs lower than for symptomatic children (P < 0.0001); differences were consistent (P < 0.0001) across all four age brackets. These differences were consistent across all institutions and by sex, ethnicity, and race. Asymptomatic children with diabetes (odds ratio [OR], 6.5; P = 0.01), a recent contact (OR, 2.3; P = 0.02), and testing for surveillance (OR, 2.7; P = 0.005) had higher estimated risks of having a Ct value in the lowest quartile than children without, while an immunocompromised status had no effect. Children with asymptomatic SARS-CoV-2 infection had lower levels of virus in their nasopharynx/oropharynx than symptomatic children, but the timing of infection relative to diagnosis likely impacted levels in asymptomatic children. Caution is recommended when choosing diagnostic tests for screening of asymptomatic children.

Plasma Metagenomic Next-Generation Sequencing Assay for Identifying Pathogens: a Retrospective Review of Test Utilization in a Large Children's Hospital.

Plasma metagenomic next-generation sequencing (mNGS) is a new diagnostic method used to potentially identify multiple pathogens with a single DNA-based diagnostic test. The test is expensive, and little is understood about where it fits into the diagnostic schema. We describe our experience at Texas Children's Hospital with the mNGS assay by Karius from Redwood City, CA, to determine whether mNGS offers additional diagnostic value when performed within 1 week before or after conventional testing (CT) (i.e., concurrently). We performed a retrospective review of all patients who had mNGS testing from April to June of 2019. Results for mNGS testing, collection time, time of result entry into the electronic medical record, and turnaround time were compared to those for CT performed concurrently. Discordant results were further reviewed for changes in antimicrobials due to the additional organism(s) identified by mNGS. Sixty patients had mNGS testing; the majority were immunosuppressed (62%). There was 61% positive agreement and 58% negative agreement between mNGS and CT. The mean time of result entry into the electronic medical record for CT was 3.5 days earlier than the mean result time for mNGS. When an additional organism(s) was identified by mNGS, antimicrobials were changed 26% of the time. On average, CT provided the same result as mNGS, but sooner than mNGS. When additional organisms were identified by mNGS, there was no change in management in the majority of cases. Overall, mNGS added little diagnostic value when ordered concurrently with CT.

Coronavirus Disease 2019 in Children Cared for at Texas Children's Hospital: Initial Clinical Characteristics and Outcomes.

We describe the clinical course of 57 children with coronavirus disease 2019 (COVID-19) cared for through a single hospital system. Most children were mildly symptomatic, and only a few patients with underlying medical conditions required hospitalization. Systemwide patient evaluation processes allowed for prompt identification and management of patients with COVID-19.

Neonatal Bacteremia and Cutaneous Lesions Caused by Photorhabdus luminescens: A Rare Gram-Negative Bioluminescent Bacterium.

Photorhabdus luminescens is a rare bacterium that causes human disease. In this report, we describe the case of a neonate with Photorhabdus luminescens bacteremia, including clinical presentation and treatment; we also report a literature review of rare human diseases.

The "Ideal" Body Weight for Pediatric Gentamicin Dosing in the Era of Obesity: A Population Pharmacokinetic Analysis.

BACKGROUND: Obese pediatric patients often require dose reductions when initiating gentamicin therapy. An appropriate method for calculating ideal body weight for dosing gentamicin in pediatric patients has not been validated. METHODS: A retrospective population pharmacokinetic study was designed and included non-intensive care pediatric patients who received gentamicin and had serum gentamicin concentrations sampled. Actual body weight (ABW), adjusted body weight, and fat-free mass (FFM) were used to describe the pharmacokinetic variables. Descriptive statistical methods were used for the population, and pharmacokinetic analysis occurred with NONMEM (ICON Plc, Dublin, Ireland). Simulation was performed to estimate dosing based on adjustments in body weight. RESULTS: A total of 520 patients met inclusion criteria (male 57.3%, mean age 9.6 ± 4.9 years, ABW 38.0 ± 24.3 kg). Obesity was present in 21.3% of the patients and overweight in 15.8%. Gentamicin was administered at 2.17 ± 0.86 mg/kg per dose. A median of 2 (interquartile range, 1-3) gentamicin serum concentrations were sampled at a median 1.8 (interquartile range, 1.1-7.8) hours after a dose. Population pharmacokinetic analysis demonstrated a 2-compartment model with allometrically scaled FFM providing the best fit. Other significant covariates included serum creatinine and age. Simulation demonstrated increased doses per body weight for traditional and once-daily dosing when using FFM for gentamicin dosing. CONCLUSIONS: FFM should be used to adjust ABW for empirically dosing gentamicin in pediatric patients aged 2-18 years.

Analysis of Invasive Community-Acquired Methicillin-Susceptible Staphylococcus aureus Infections During a Period of Declining Community Acquired Methicillin-Resistant Staphylococcus aureus Infections at a Large Children's Hospital.

BACKGROUND: The epidemiology of community acquired (CA) Staphylococcus aureus infections is changing in the United States. We investigated the current epidemiology of S. aureus infections at Texas Children's Hospital. METHODS: Patients with CA-S. aureus skin and soft tissue and invasive infections were retrospectively identified from January 1, 2007 to December 31, 2014. Invasive CA-MSSA isolates were characterized by pulsed field gel electrophoresis, Spa typing, agr type and presence of lukSF-PV (pvl) genes. Medical records were reviewed. Statistical analyses included Fisher exact, χ for trend and Wilcoxon tests. RESULTS: CA-MRSA infections decreased by 60.4% (1461-578 infections) from 2007 to 2014 (P < 0.0001), while CA-MSSA infections averaged 550 infections annually. Invasive CA-MRSA infections decreased by 67.2% from 61 to 20 infections (P < 0.0001); invasive CA-MSSA averaged 44 infections annually. Among 296 invasive CA-MSSA isolates, 74 (25%) isolates were USA300 and 88 (30%) were pvl+. USA300 declined among invasive CA-MSSA over time (P < 0.008). Musculoskeletal infections were most common (242/296, 82%); 52/242 (21.5%) isolates were USA300 and 62/242 (25.6%) pvl+. All 18 isolates from musculoskeletal infections with deep venous thrombosis and/or septic shock were pvl+ and 16/18 (88.9%) were USA300. Pneumonia isolates were mainly USA300 (8, 66.7%) and pvl+ (11, 91.7%). CONCLUSIONS: MSSA now cause the majority of invasive CA-S. aureus infections at our institution. Molecular analysis of invasive CA-MSSA isolates suggests strain diversity with USA300 on the decline and that disease presentations are to some extent strain specific. Changes in the CA-S. aureus epidemiology may, in part, be related to changes in immunity to the USA300 clone in the general population.

An Outbreak of Burkholderia cepacia Complex Infections Associated with Contaminated Liquid Docusate.

OBJECTIVE To investigate an outbreak of Burkholderia cepacia complex and describe the measures that revealed the source. SETTING A 629-bed, tertiary-care, pediatric hospital in Houston, Texas. PATIENTS Pediatric patients without cystic fibrosis (CF) hospitalized in the pediatric and cardiovascular intensive care units. METHODS We investigated an outbreak of B. cepacia complex from February through July 2016. Isolates were evaluated for molecular relatedness with repetitive extragenic palindromic polymerase chain reaction (rep-PCR); specific species identification and genotyping were performed at an independent laboratory. The investigation included a detailed review of all cases, direct observation of clinical practices, and respiratory surveillance cultures. Environmental and product cultures were performed at an accredited reference environmental microbiology laboratory. RESULTS Overall, 18 respiratory tract cultures, 5 blood cultures, 4 urine cultures, and 3 stool cultures were positive in 24 patients. Among the 24 patients, 17 had symptomatic infections and 7 were colonized. The median age of the patients was 22.5 months (range, 2-148 months). Rep-PCR typing showed that 21 of 24 cases represented the same strain, which was identified as a novel species within the B. cepacia complex. Product cultures of liquid docusate were positive with an identical strain of B. cepacia complex. Local and state health departments, as well as the CDC and FDA, were notified, prompting a multistate investigation. CONCLUSIONS Our investigation revealed an outbreak of a unique strain of B. cepacia complex isolated in clinical specimens from non-CF pediatric patients and from liquid docusate. This resulted in a national alert and voluntary recall by the manufacturer. Infect Control Hosp Epidemiol 2017;38:567-573.

Syphilis during pregnancy: a preventable threat to maternal-fetal health.

Syphilis remains the most common congenital infection worldwide and has tremendous consequences for the mother and her developing fetus if left untreated. Recently, there has been an increase in the number of congenital syphilis cases in the United States. Thus, recognition and appropriate treatment of reproductive-age women must be a priority. Testing should be performed at initiation of prenatal care and twice during the third trimester in high-risk patients. There are 2 diagnostic algorithms available and physicians should be aware of which algorithm is utilized by their testing laboratory. Women testing positive for syphilis should undergo a history and physical exam as well as testing for other sexually transmitted infections, including HIV. Serofast syphilis can occur in patients with previous adequate treatment but persistent low nontreponemal titers (<1:8). Syphilis can infect the fetus in all stages of the disease regardless of trimester and can sometimes be detected with ultrasound >20 weeks. The most common findings include hepatomegaly and placentomegaly, but also elevated peak systolic velocity in the middle cerebral artery (indicative of fetal anemia), ascites, and hydrops fetalis. Pregnancies with ultrasound abnormalities are at higher risk of compromise during syphilotherapy as well as fetal treatment failure. Thus, we recommend a pretreatment ultrasound in viable pregnancies when feasible. The only recommended treatment during pregnancy is benzathine penicillin G and it should be administered according to maternal stage of infection per Centers for Disease Control and Prevention guidelines. Women with a penicillin allergy should be desensitized and then treated with penicillin appropriate for their stage of syphilis. The Jarisch-Herxheimer reaction occurs in up to 44% of gravidas and can cause contractions, fetal heart rate abnormalities, and even stillbirth in the most severely affected pregnancies. We recommend all viable pregnancies receive the first dose of benzathine penicillin G in a labor and delivery department under continuous fetal monitoring for at least 24 hours. Thereafter, the remaining benzathine penicillin G doses can be given in an outpatient setting. The rate of maternal titer decline is not tied to pregnancy outcomes. Therefore, after adequate syphilotherapy, maternal titers should be checked monthly to ensure they are not increasing four-fold, as this may indicate reinfection or treatment failure.

Prevalence of co-infection between respiratory syncytial virus and influenza in children.

BACKGROUND: Respiratory syncytial virus (RSV) and influenza have varying degree of seasonal overlap. OBJECTIVE: To determine the prevalence of co-infection of RSV and influenza compared to the prevalence of those infections independently when both are in season. METHODS: This was a retrospective cross-sectional study of children evaluated between July 2010 and June 2013 for viral respiratory infection using multiplex PCR. Seasonality was defined retrospectively as weeks when >2% of the total annual positive tests were obtained and was calculated for influenza A, influenza B, and RSV independently. Periods of overlapping seasonality of RSV and influenza A and RSV and influenza B were identified. The expected incidences of co-infection were modeled as the product of the incidences of the individual viruses. RESULTS: 13,664 specimens were sent for PCR during the study period. Over all 3 seasons, RSV overlapped with influenza A and B for 22 and 18weeks, respectively; in 2011-12, RSV overlapped with neither influenza A nor B. Based on modeling, there were 6-7 fold fewer cases of RSV/influenza co-infection observed than expected: RSV/influenza A 77 vs. 12, (p≤0.001; RSV/influenza B 76 vs. 11 (p≤0.001). CONCLUSIONS: The observed incidence of co-infectivity of RSV and influenza was significantly less than the expected incidence even when both were co-circulating. In light of these data, it may be reasonable to forgo rapid influenza testing or empiric antiviral treatment for children whom rapid RSV testing is positive and who are at low risk of influenza-related complications, especially in times of antiviral therapy shortages.

Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children.

Diagnosis of tuberculosis in children is challenging; even with advanced technologies, the diagnosis is often difficult to confirm microbiologically in part due to the paucibacillary nature of the disease. Clinical diagnosis lacks standardization, and traditional and molecular microbiologic methods lack sensitivity, particularly in children. Immunodiagnostic tests may improve sensitivity, but these tests cannot distinguish tuberculosis disease from latent infection and some lack specificity. While molecular tools like Xpert MTB/RIF have advanced our ability to detect Mycobacterium tuberculosis and to determine antimicrobial resistance, decades old technologies remain the standard in most locales. Today, the battle against this ancient disease still poses one of the primary diagnostic challenges in pediatric laboratory medicine.

Performance of a parallel diagnostic algorithm for HIV diagnosis in low risk pediatric and obstetric patient populations.

BACKGROUND: Little is known about the clinical performance of the 2010 APHL/CDC Western-blot independent HIV testing algorithm in low risk pediatric and obstetric patients. OBJECTIVE: To evaluate the performance of an alternate Western-blot independent algorithm and the individual algorithm components in diagnosing HIV infections in low risk pediatric and obstetric patients. STUDY DESIGN: 6242 specimens from pediatric and obstetric patients were tested by the Bio-Rad Multispot HIV-1/HIV-2 (MS) and VITROS Anti HIV 1+2 (VITROS) assays. 913 specimens were also tested by the ARCHITECT HIV Ag/Ab Combo assay (ARCHITECT). Discordant specimens were tested by the APTIMA HIV-1 RNA qualitative assay (RNA Qual). RESULTS: Twenty-eight specimens tested positive for HIV-1 by both MS and VITROS, 4 of these 28 specimens were also tested by and positive by ARCHITECT; all 28 positives identified by the algorithm were positive by viral load analysis. MS identified 164 preliminary positives, which were not confirmed as true positives, representing a specificity of 97.4%. This specificity varied between patient populations (96.1% in the pediatric population and 99.1% in the obstetric population). The specificities of VITROS and ARCHITECT were 99.2% and 99.4% for pediatric patients; 99.7% and 99.8% for obstetric patients, respectively. CONCLUSION: Our results highlight suboptimal specificity of MS in pediatric patients, and a lower specificity in both pediatric and obstetric patients relative to either VITROS or ARCHITECT. Additionally, parallel testing with both a third and fourth generation EIA in a low risk patient population provides a potential alternative to Western-blot dependent algorithms for confirmation.

Polymicrobial bloodstream infections in the neonatal intensive care unit are associated with increased mortality: a case-control study.

BACKGROUND: Polymicrobial infections in adults and children are associated with increase in mortality, duration of intensive care and healthcare costs. Very few studies have characterized polymicrobial bloodstream infections in the neonatal unit. Considerable variation has been reported in incidence of polymicrobial infections and associated clinical outcomes. We characterized the risk factors and outcomes of polymicrobial bloodstream infections in our neonatal units in a tertiary hospital in North America. METHODS: In a retrospective case control study design, we identified infants in the neonatal intensive care unit with positive blood cultures at Texas Children's Hospital, over a 16-year period from January 1, 1997 to December 31, 2012. Clinical data from online databases were available from January 2009 to December 2012. For each polymicrobial bloodstream infection (case), we matched three infants with monomicrobial bloodstream infection (control) by gestational age and birth weight. RESULTS: We identified 2007 episodes of bloodstream infections during the 16 year study period and 280 (14%) of these were polymicrobial. Coagulase-negative Staphylococcus, Enterococcus, Klebsiella and Candida were the most common microbial genera isolated from polymicrobial infections. Polymicrobial bloodstream infections were associated with more than 3-fold increase in mortality and an increase in duration of infection. Surgical intervention was a significant risk factor for polymicrobial infection. CONCLUSION: The frequency and increased mortality emphasizes the clinical significance of polymicrobial bloodstream infections in the neonatal intensive care unit. Clinical awareness and focused research on neonatal polymicrobial infections is urgently needed.

Real-time polymerase chain reaction detection of asymptomatic Clostridium difficile colonization and rising C. difficile-associated disease rates.

OBJECTIVE: To evaluate the accuracy of real-time polymerase chain reaction (PCR) for Clostridium difficile-associated disease (CDAD) detection, after hospital CDAD rates significantly increased following real-time PCR initiation for CDAD diagnosis. DESIGN: Hospital-wide surveillance study following examination of CDAD incidence density rates by interrupted time series design. SETTING: Large university-based hospital. PARTICIPANTS: Hospitalized adult patients. METHODS: CDAD rates were compared before and after real-time PCR implementation in a university hospital and in the absence of physician and infection control practice changes. After real-time PCR introduction, all hospitalized adult patients were screened for C. difficile by testing a fecal specimen by real-time PCR, toxin enzyme-linked immunosorbent assay, and toxigenic culture. RESULTS: CDAD hospital rates significantly increased after changing from cell culture cytotoxicity assay to a real-time PCR assay. One hundred ninety-nine hospitalized subjects were enrolled, and 101 fecal specimens were collected. C. difficile was detected in 18 subjects (18%), including 5 subjects (28%) with either definite or probable CDAD and 13 patients (72%) with asymptomatic C. difficile colonization. CONCLUSIONS: The majority of healthcare-associated diarrhea is not attributable to CDAD, and the prevalence of asymptomatic C. difficile colonization exceeds CDAD rates in healthcare facilities. PCR detection of asymptomatic C. difficile colonization among patients with non-CDAD diarrhea may be contributing to rising CDAD rates and a significant number of CDAD false positives. PCR may be useful for CDAD screening, but further study is needed to guide interpretation of PCR detection of C. difficile and the value of confirmatory tests. A gold standard CDAD diagnostic assay is needed.

Clinical evaluation of a real-time PCR assay for identification of Salmonella, Shigella, Campylobacter (Campylobacter jejuni and C. coli), and shiga toxin-producing Escherichia coli isolates in stool specimens.

Enteric illness affects millions of individuals annually in the United States and results in >50,000 hospitalizations. The rapid and accurate identification of bacterial pathogens associated with gastroenteritis can aid acute patient management decisions, including the use of antibiotic therapy and infection control. This study compared the ProGastro SSCS multiplex real-time PCR assay (Gen-Probe Prodesse, San Diego, CA) to culture for the identification of Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Salmonella spp., and Shigella spp. and to broth enrichment followed by an FDA-cleared enzyme immunoassay (EIA) for the identification of Shiga toxin-producing Escherichia coli (STEC) isolates in stool specimens. Stool samples submitted in preservatives for routine culture and EIA were prospectively enrolled and tested at four clinical centers. Discrepancies between the ProGastro SSCS assay and culture or EIA were resolved using bidirectional sequencing. The overall prevalence of the pathogens as detected by culture was 5.6% (1.8% Campylobacter, 1.8% Salmonella, 1.3% Shigella, and 0.8% STEC). When results based on the ProGastro SSCS assay and bidirectional sequencing were applied, the overall prevalence increased to 8.3% (2.3% Campylobacter, 2.6% Salmonella, 1.8% Shigella, and 1.6% STEC). Following resolution of the discrepant results, the sensitivity of the ProGastro SSCS assay was 100% for all pathogens, and the specificities ranged from 99.4% to 100%. The sensitivity of culture compared to sequence-confirmed ProGastro SSCS results ranged from 52.9% to 76.9%, with the specificities ranging from 99.9% to 100%. Overall, these results suggest that the ProGastro SSCS assay is highly sensitive and specific in a clinical setting.