Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5′-untranslated region (5′-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

Burden of Intestinal Parasitic Infections among Children from Five Schools in Bhairahawa, Nepal: A Comparative Cross-Sectional Study

INTRODUCTION: Intestinal parasites cause significant morbidity and mortality, particularly in the tropics including Nepal. The main objective of this study was to explore the burden of intestinal parasitic infections among children in 5 different primary schools in Bhairahawa, Nepal. MATERIAL AND METHODS: This was a cross-sectional comparative study among 408 children from 5 schools in Bhirahawa, Nepal. Stool specimens collected in a plastic container were transported to microbiology laboratory in Universal College of Medical Sciences Teaching Hospital (UCMSTH) immediately. Each sample was examined macroscopically and microscopically for the evidence of parasitic infection. All samples were re-analysed by sedimentation and floatation concentration techniques one after another. RESULTS: The overall prevalence of the parasitic infection was 46.5%. The prevalence varied by the methods that included routine microscopy (23.2%), sedimentation (41.6%) and flotation (8.3%). Ascaris lumbricoides was the most common (29.1%) parasite followed by Entamoeba histolytica (6.1%). Mixed infection was also seen in 7.8% of the samples. Factors such as children’s academic year, age, religion, existing illness, household water sources, meat consumed, domestic animals at house, and recent history of taking anti-helminthics were significantly associated with the intestinal parasitic infections. Prevalence of intestinal parasitic infection was higher in public school (61.1%) compared to private school (37%). Male students had slightly more infections (47.3%) than females (45.7%). CONCLUSIONS: Enteric parasitic infection was very high among the primary schools’ students in Bhairahawa, Nepal. Integrating concentration techniques in routine test can help to detect most of the enteric parasites in stool specimens.

Molecular diversity survey on diarrheagenic Escherichia coli isolates among children with gastroenteritis in Fars, Iran

Aim: To differentiate Escherichia coli isolates from diarrheal pediatric patients in clinical laboratories. Materials & methods: Patients with watery diarrhea were selected for sampling and tested for Diarrheagenic E. coli (DEC) by API kit. DEC isolates were tested for phylotyping, pathotyping and presence of determined virulence-encoding genes by specific molecular methods. Results: About 50% of isolates were detected as DECs (>55 and >31% were categorized B2 and D phylotypes respectively). Enterotoxigenic E. coli was the most and Enteroinvasive E. coli was the lowest prevalent pathotypes. csg and fim genes were the most present virulence factors. Conclusion: Typing of E. coli isolates from stool specimens will help to determine the diversity of diarrheal pathogens and take proper decisions to reduce the health burden of diarrheal diseases.

Cutaneous Granulomatosis Revealing Whipple’s Disease: Value of Tropheryma whipplei Polymerase Chain Reaction Assay for the Diagnosis

Whipple’s Disease is a rare systemic infectious disease caused by the ubiquitous actinomycetes Tropheryma whipplei (T. whipplei). We report herein a rare case of a cutaneous granulo matosis with hypercalcemia as an unusual presenting feature of Whipple’s disease. The diagnosis of the bacteria was obtained from skin and inguinal lymph node biopsy (16 rDNA PCR screening and histological examination using PAS staining). T. whipplei was also identified on saliva and stool specimens, using specific PCR and colonic biopsies. Treatment with hydroxychloroquine and doxycycline allowed a rapid resolution of symptoms with a complete recovery.

756. Clostridioides difficile Burden of Disease: A Prospective Population-Based Surveillance Study of Hospitalized Adults in Louisville, Kentucky

Background Clostridioides difficile (C. difficile) is an important cause of morbidity and mortality. C. difficile infection (CDI) may be frequently under-diagnosed because laboratory confirmation requires collection of a stool specimen from a patient with diarrhea and appropriate laboratory testing. Methods A prospective population-based CDI surveillance study was launched in 8 adult hospitals in Louisville, Kentucky on September 16, 2019. Surveillance officers in each hospital identified all cases of new-onset diarrhea (≥3 loose stools in the past but not preceding 24 hours) in Louisville residents ≥50 years of age. After informed consent, stool samples were collected and tested at the University of Louisville reference laboratory for 1) glutamate dehydrogenase (GDH) and 2) Clostridioides difficile toxins A and B using C. DIFF QUIK CHEK COMPLETE®, Techlab. We defined CDI as GDH positive and toxin positive. The study was paused on April 3, 2020, due to COVID-19 restrictions. Results There were 85,719 eligible patient-days during the study period. A total of 1541 patients had new-onset diarrhea corresponding to 1.8 cases of new-onset diarrhea per 100 eligible patient-days. We enrolled 84% (1291/1541) of patients with new-onset diarrhea and tested stool samples for C. difficile from 82% (1055/1291) for a testing density of 123 per 10,000 patient-days. Of the 1055 tested stool specimens, 73 (7%) were GDH positive and toxin positive (Figure 1) yielding a hospital-based CDI incidence of 8.5 CDI cases per 10,000 patient-days. Figure 1. Patient Ascertainment Flow Chart Conclusion New-onset diarrhea was common among hospitalized adults ≥50 years of age. CDI was frequently identified through stool specimens collected from eligible inpatients with new-onset diarrhea. Further analysis of these data and additional laboratory testing will contribute to a better understanding of the frequency of CDI underdiagnosis and the burden of CDI in the United States. Disclosures Ruth Carrico, PhD, DNP, APR, CIC, Pfizer (Consultant, Research Grant or Support, Speaker's Bureau)Sanofi Pasteur (Consultant, Grant/Research Support, Speaker's Bureau) Fredrick J. Angulo, DVM, PhD, Pfizer, Inc. (Employee) Joann Zamparo, MPH, Pfizer, Inc. (Employee) Elisa Gonzalez, MS, Pfizer, Inc. (Employee) Kimbal D. Ford, PharmD, Pfizer, Inc. (Employee) Julio Ramirez, M.D., FACP, Pfizer, Inc. (Scientific Research Study Investigator, Research Grant or Support, Speaker's Bureau)

749. A Nurse-Driven Protocol for Early Detection of Clostridiodes difficile Infections

Background Clostridiodes difficile infections (CDI) are a significant cause of hospital acquired infections, resulting in significant morbidity and mortality. Early detection of CDI has been shown to reduce the spread of CDI within the hospital. As nurses are frequently at the patient’s bedside, we proposed to empower the nursing staff to assess, collect stool samples, and order C. difficile testing. Methods Rates of CDI were measured by our Infection Control Department. Hospital-onset CDI (HO-CDI) was defined as a positive C. difficile PCR assay after 3 days of admission, defined as a stay of at least 3 midnights. Community-onset CDI (CO-CDI) was defined as any case that was diagnosed in the Emergency Department or inpatient ward < 3 days of hospitalization based on stool testing as above. Nursing was instructed and empowered to assess, collect stool specimens, and place an order for C. difficile testing, based on the criteria of ≥3 loose or watery stools over 24 hours. Nursing was also educated to not order a test if patients had received stool softeners, enemas, or laxatives within 24 hours. The protocol was initiated in February 2019. Results Rates of HO-CDI increased during the intervention period, rising from 2.6 cases/10000 patient days and peaking at 17.7 cases/10000 patient days (average 6.7 vs. 12.1 monthly cases per 10,000 patient days. Rates of CO-CDI did not significantly change (12.4 vs. 11.5 monthly cases per 10000 patient days). Due to concerns of inappropriate testing, which included testing after laxatives, enemas, or sending specimens despite < 3 stools over 24 hours, the protocol was discontinued in June 2019. Although the HO-CDI rate remained elevated over the next month, the rate subsequently decreased over the next several months (12.1 vs. 8.0 cases per 10000 patient days). Overall testing also increased over the study period (148.3 vs. 169.9 cases/per 10000 patient days).Figure 1 - Clostridiodes difficile rates Figure 2 - CDI testing rates Conclusion A nursing driven protocol resulted in increased HO-CDI and overall CDI rates suggesting that the intervention may have been a factor in increasing the frequency of HO-CDI diagnoses, although the possibility of misdiagnosis of colonization for true CDI cannot be excluded. Further education of nursing staff may be a potential intervention in improving appropriate CDI testing. Disclosures All Authors: No reported disclosures

1296. Genomic Analysis of Human-Associated Outbreak Caused by Salmonella enterica serovar Infantis in Cali, Colombia

Background Salmonella enterica serovar Infantis is emerging as a leading cause of foodborne infections. The Colombian National Health Institute reported 263 Infantis among all received Salmonella(n=12,966) from 1997 to 2018 being the 5th most frequent serovar in human clinical samples during this period. In this study, Infantis outbreak-associated isolates were sequenced and collectively analysed with global strains from different sources to compare their genomic content and phylogeny Figure 1. Genomic Comparisons Methods Between September 15-20 of 2019, 160 patients with gastroenteric symptoms resulting from the ingestion of a common food, arrived at Fundación Valle del Lilí. Salmonella spp were cultured from stool specimens of patients and 15 isolates were sent to the Genomic Unit of Agrosavia for whole-genome sequencing. Genomic DNA was sequenced using an Illumina®MiSeqX10 platform. Genome assembly was performed with standardized bioinformatics pipelines and in-silico serotyping with SISTR. Genomic comparisons included newly-sequenced isolates of Infantis(n=6) from a Colombian poultry farms as well as datasets from Patric(n=441) and EnteroBase(n=54). Results The 15 outbreak isolates were identified by in silico serotyping as S. Infantis, these isolates showed phenotypic sensitivity to all tested antibiotics except by tetracycline. Antimicrobial resistance plasmids IncFIBKpn3, IncX4 and the host-environment persistence plasmid pSL483 were only detected in 2 outbreak-related and 1 in poultry isolates. Out of a total of 511 high-quality sequences, ST32 was the most prevalent and were phylogenetically grouped into a single non-host specific clade. Outbreak-isolates were included in a monophyletic group with some genomes from the US and Chile, suggesting that the parent strain could have originated in those countries. Conclusion The magnitude of the outbreak(288 informed-cases) evidenced a high-virulent potential of outbreak-isolates. Routine sequencing of S.enterica and availability of genomes in Colombia can improve outbreak detection and resolution in the future. The presence of plasmids, although with low frequency, suggests a risk of the appearance of resistant clones. Disclosures All Authors: No reported disclosures

Draft Genome Sequences of Multidrug-Resistant Shigella Strains Isolated from Diarrheal Patients in Bangladesh

The emergence of multidrug-resistant (MDR) Shigella strains has impaired the efficacy of first-line antimicrobials and exacerbated diarrhea-associated morbidity and mortality worldwide. We report the draft genome sequences of 11 MDR Shigella strains isolated from the stool specimens of diarrheal patients in Bangladesh.