746. Clinical and Microbiological Characteristics of Clostridioides difficile Infection After a Change in Diagnostic Testing Algorithm

Background There is a lack of consensus on the most appropriate testing for C. difficile infection (CDI). The objective of this study was to determine the clinical and microbiological characteristics of CDI following a switch from stool PCR testing only to PCR reflexed to toxin testing. Figure 1. PCR Cycle Threshold Values Methods We reviewed the characteristics and outcomes of 50 consecutive patients who tested positive for CD by PCR (Xpert CD, Cepheid) between October 2019 and January 2020 at the Hines VA Hospital. Cases were defined by results of reflex toxin testing (Cdiff quick check complete, Alere/TechLab) after a positive PCR result. Baseline characteristics, symptoms, initial laboratory data, and treatments were compared as well as patient outcomes, including hospital readmission due to CDI at 30 days, and recurrent CDI (rCDI) at 30 and 90 days. The cycle threshold for the stool PCR result was recorded. Stools were cultured anaerobically for CD, and restriction endonuclease analysis (REA) strain typing was performed on the recovered CD isolates. Results Toxin testing was positive in 19/50 (38%) cases. Compared to stool toxin-negative cases, toxin-positive cases were older (95% vs. 71% were age ≥ 65, p = 0.06), more likely to have a history of CDI (37% vs. 23%, p = 0.34), and have ≥ 1 CDI episodes within 6 months (37% vs. 19%, p = 0.26). Treatment for CDI was more common in patients who had a positive toxin text. (95% vs 61%, p= 0.009). Among the 38 patients that received treatment, 33 received vancomycin (87%) and 8 patients (21%) had rCDI at 30 days. Of the 8 patients with rCDI, 2 were re-admitted to the hospital for CDI. The average PCR cycle threshold was lower in the toxin-positive stools compared to toxin-negative stools (24.46 and 29.96, p< 0.001; Fig. 1) The endemic REA group Y was the most common CD strain recovered (30%) and the previously epidemic and virulent REA group BI strain was recovered in 11% of the cases. Conclusion CDI cases diagnosed by positive stool PCR and positive toxin tests had more typical risk factors for CDI, a lower PCR cycle threshold and were more likely to have been treated for CDI. Outcomes were similar in this setting where infection with the virulent BI strain was uncommon. Disclosures Stuart Johnson, MD, Acurx Pharmaceuticals (Advisor or Review Panel member)Bio-K+ (Advisor or Review Panel member)Ferring Pharmaceutical (Advisor or Review Panel member)

Human Adenovirus Type 7 Infections in Hubei, China During 2018-2019: Epidemic Features and Genetic Characterization of the Detected Viruses

Human adenoviruses (HAdVs) type 7 can cause severe respiratory disease. During the period between December 2018 and August 2019, HAdV-7 infection was identified in 129 patients in Wuhan Children’s Hospital, Hubei Province, China. Samples were collected from hospitalized children and metagenomic sequencing was applied to detect the HAdV infections. Hemophagocytic lymphohistiocystosis (HLH) related to HAdV infections was observed in some patients clinically and patients were divided into two groups based on this to test the differences among clinical indicators. Genome variation, in silico restriction endonuclease analysis (REA), and phylogenetic analyses were carried out to show the genome characterization of HAdV-7 in this study. It was found that many indicators, such as all blood routine indicators, in patients of the HLH group showed significant levels. In this study, REA revealed that HAdV-7 might belong to genome 7d and genome variation analysis displayed the stable genome of HAdV. HAdV-7 is an ongoing threat to the public, and global surveillance should be established.

The Relative Role of Toxins A and B in the Virulence of Clotridioides difficile

Most pathogenic strains of C. difficile possess two large molecular weight single unit toxins with four similar functional domains. The toxins disrupt the actin cytoskeleton of intestinal epithelial cells leading to loss of tight junctions, which ultimately manifests as diarrhea in the host. While initial studies of purified toxins in animal models pointed to toxin A (TcdA) as the main virulence factor, animal studies using isogenic mutants demonstrated that toxin B (TcdB) alone was sufficient to cause disease. In addition, the natural occurrence of TcdA−/TcdB+ (TcdA−/B+)mutant strains was shown to be responsible for cases of C. difficile infection (CDI) with symptoms identical to CDI caused by fully toxigenic (A+/B+) strains. Identification of these cases was delayed during the period when clinical laboratories were using immunoassays that only detected TcdA (toxA EIA). Our hospital laboratory at the time performed culture as well as toxA EIA on patient stool samples. A total of 1.6% (23/1436) of all clinical isolates recovered over a 2.5-year period were TcdA−/B+ variants, the majority of which belonged to the restriction endonuclease analysis (REA) group CF and toxinotype VIII. Despite reports of serious disease due to TcdA−/B+ CF strains, these infections were typically mild, often not requiring specific treatment. While TcdB alone may be sufficient to cause disease, clinical evidence suggests that both toxins have a role in disease.

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (MAP) in dairy cattle of Nepal

Paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an economically important, endemic in many parts of the globe, and regarded as high prevalent disease of domestic and wild animals, especially ruminants, which is manifest as chronic granulomatous enteritis with decreased milk production, with serious cases resulting in progressive emaciation and death. Understanding the genetic variability of MAP, strains are important in diagnosis, epidemiological investigation, and therefore the formation of strategies for prevention and control of the disease. Thus, this study was designed to grasp the molecular characterization of MAP isolates of Nepal, as pioneer research of this area. Total of 46 MAP isolates obtained from cattle population of three different locations of dairy pocket areas of Chitwan, Nepal were typed using IS1311 polymerase chain reaction-restriction endonuclease analysis (PCR-REA) to research the MAP genotype of Nepal. The extracted DNA samples (n=46) were analyzed for the presence of MAP specific sequences (IS900) using PCR and DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. All the DNA samples were positive for the entire three MAP specific sequences based PCRs. This study revealed that ‘Bison type’ strain is the single most prevalent MAP genotype circulating within the domestic cattle population of Nepal. IS1311 PCR-REA showed that MAP DNA samples of Nepal origin belonged to ‘Bison type’, whereas, IS1311 L2 PCR-REA method showed similarity with "Indian Bison type" and different restriction profiles of ‘Bison type’ genotype as compared to non-Indian strains. The study concludes that in Nepal, "Bison type" MAP stains was prevalent in all the MAP samples obtained from dairy cattle. These results have important epidemiological implications regarding control and prevention of paratuberculosis in Nepal.

Characterization of Clostridioides difficile isolates recovered from two Phase 3 surotomycin treatment trials by restriction endonuclease analysis, PCR ribotyping and antimicrobial susceptibilities

Objectives To investigate the molecular epidemiology and antimicrobial susceptibility of Clostridioides difficile isolates from patients with C. difficile infection (CDI) from two Phase 3 clinical trials of surotomycin. Methods In both trials [Protocol MK-4261-005 (NCT01597505) conducted across Europe, North America and Israel; and Protocol MK-4261-006 (NCT01598311) conducted across North America, Asia-Pacific and South America], patients with CDI were randomized (1:1) to receive oral surotomycin (250 mg twice daily) or oral vancomycin (125 mg four times per day) for 10 days. Stool samples were collected at baseline and C. difficile isolates were characterized by restriction endonuclease analysis (REA) and PCR ribotyping. Susceptibility testing was performed by agar dilution, according to CLSI recommendations. Results In total, 1147 patients were included in the microbiological modified ITT population. Of 992 recovered isolates, 922 (92.9%) were typed. There was a high association between REA groups and their corresponding predominant PCR ribotype (RT) for BI, DH, G and CF strains. REA group A showed more diverse PCR RTs. Overall, the most common strain was BI/RT027 (20.3%) followed by Y/RT014/020 (15.0%) and DH/RT106 (7.2%). The BI/RT027 strain was particularly prevalent in Europe (29.9%) and Canada (23.6%), with lower prevalence in the USA (16.8%) and Australia/New Zealand (3.4%). Resistance was most prevalent in the BI/RT027 strain, particularly to metronidazole, vancomycin and moxifloxacin. Conclusions A wide variation in C. difficile strains, both within and across different geographical regions, was documented by both REA and ribotyping, which showed overall good correlation.

Efficacy of Bezlotoxumab in Trial Participants Infected With Clostridioides difficile Strain BI Associated With Poor Outcomes

Background Bezlotoxumab reduced rates of recurrent Clostridioides difficile infection (rCDI) vs placebo in Monoclonal Antibodies for C. difficile Therapy (MODIFY) I/II trial participants receiving antibacterial drug treatment for CDI. A secondary objective of MODIFY I/II was to assess bezlotoxumab’s efficacy against C. difficile strains associated with increased rates of morbidity and mortality. Methods In this post-hoc analysis of pooled MODIFY I/II data, efficacy endpoints were assessed in participants infected with restriction endonuclease analysis BI and non-BI strains of C. difficile at study entry. Treatment outcomes were compared between participants receiving bezlotoxumab (alone or with actoxumab [B, B+A]) and those receiving no bezlotoxumab (placebo or actoxumab [P, A]). Results From 2559 randomized participants, C. difficile was isolated from 1588 (67.2%) baseline stool samples. Participants with BI strains (n = 328) were older and had more risk factors for rCDI than non-BI strain participants (n = 1260). There were no differences in initial clinical cure rate between BI and non-BI strains in either group. The rCDI rate for BI strains treated with bezlotoxumab was lower than for the no bezlotoxumab group (B, B+A vs P, A: 23.6% vs 43.9%) and was also lower for the non-BI strains (B, B+A vs P, A: 21.4% vs 36.1%). Rates of 30-day CDI-associated rehospitalization were greater with BI vs non-BI strains in both groups. Conclusions Infection with BI strains of C. difficile predicted poor outcomes in the MODIFY I/II trials. Bezlotoxumab (alone or with actoxumab) treatment was effective both in BI and non-BI subpopulations.

Sequential introduction of a multistep testing algorithm and nucleic acid amplification testing leading to an increase in Clostridioides difficile detection and a trend toward increased strain diversity

Background:Most clinical microbiology laboratories have replaced toxin immunoassay (EIA) alone with multistep testing (MST) protocols or nucleic acid amplification testing (NAAT) alone for the detection of C. difficile.Objective:Study the effect of changing testing strategies on C. difficile detection and strain diversity.Design:Retrospective study.Setting:A Veterans’ Affairs hospital.Methods:Initially, toxin EIA testing was replaced by an MST approach utilizing a glutamate dehydrogenase (GDH) and toxin EIA followed by tcdB NAAT for discordant results. After 18 months, MST was replaced by a NAAT-only strategy. Available patient stool specimens were cultured for C. difficile. Restriction endonuclease analysis (REA) strain typing and quantitative in vitro toxin testing were performed on recovered isolates.Results:Before MST (toxin EIA), 79 of 708 specimens (11%) were positive, and after MST (MST-A), 121 of 517 specimens (23%) were positive (P < .0001). Prior to NAAT-only testing (MST-B), 80 of the 490 specimens (16%) were positive by MST, and after NAAT-only testing was implemented, 67 of the 368 specimens (18%) were positive (P = nonsignificant). After replacing toxin EIA testing, REA strain group diversity increased (8, 13, 13, and 10 REA groups in the toxin EIA, MST-A, MST-B, and NAAT-only periods, respectively) and in vitro toxin concentration decreased. The average log10 toxin concentration of the isolates were 2.08, 1.88, 1.20 and 1.55 ng/mL for the same periods, respectively.Conclusions:MST and NAAT had similar detection rates for C. difficile. Compared to toxin testing alone, they detected increased diversity of C. difficile strains, many of which were low toxin producing.

Detection of Mycobacterium avium subsp. paratuberculosis (MAP) from Subclinical Caprine Paratuberculosis Cases of Odisha

Paratuberculosis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP) and is a chronic, intestinal tract infection in the ruminant sector globally. A total of 122 EDTA mixed blood samples, 121 serum and 16 pooled faecal samples were collected from farms of 4 different districts i.e. Nayagarh, Cuttack, Khordha and Angul and a blind review was conducted at the Animal Health Division, CIRG, Mathura. Microscopic examination of 16 pooled faecal samples revealed +2 reactivity to Acid-Fast Bacilli. All the serum samples were subjected to indirect ELISA. Out of them, 23 (19.01%), 85 (70.25%), shows strongly positive, positive, antibody titre respectively. EDTA blood samples of 23 ELISA-strongly positive were subjected to 413 bp IS900 PCR and 11 (9%) of them were found positive for Mycobacterium avium subsp. Paratuberculosis (MAP). MAP isolates were further subjected to genotyping using 608 bpIS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) and 2 (1.64%) of them matched with “Indian Bison Type”. Genotyping of the isolates using IS1311 PCR-REA revealed that goat population of Odisha are primarily infected with “Indian Bison Type” strains.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Genetic Variation and Biological Activity of Two Closely Related Alphabaculoviruses during Serial Passage in Permissive and Semi-Permissive Heterologous Hosts

Phylogenetic analyses suggest that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) may be strains of the same virus species. Most of the studies comparing their biological activities have been performed in their homologous hosts. A comparison of host range and stability in alternative hosts was performed. The host range of these viruses was compared using high concentrations of inoculum to inoculate second instars of six species of Lepidoptera. One semi-permissive host (Spodoptera littoralis) and one permissive host (S. exigua) were then selected and used to perform six serial passages involving a concentration corresponding to the ~25% lethal concentration for both viruses. Restriction endonuclease analysis showed fragment length polymorphisms in every host-virus system studied. In S. littoralis, serial passage of MbMNPV resulted in decreased pathogenicity and an increase in speed-of-kill, whereas no significant changes were detected for HearMNPV with respect to the initial inoculum. In contrast, both viruses showed a similar trend in S. exigua. These results highlight the low genetic diversity and a high phenotypic stability of HearMNPV with respect to the original inoculum after six successive passages in both insect hosts. This study concludes that host-baculovirus interactions during serial passage are complex and the process of adaptation to a novel semi-permissive host is far from predictable.