The association between phylogenetic lineage and the subclinical phenotype of pulmonary tuberculosis: A retrospective 2-cohort study.

BACKGROUND: Subclinical pulmonary tuberculosis (PTB) is an asymptomatic disease state between established TB infection and symptomatic (clinical) TB disease. It is present in 20-25% of PTB patients in high-income countries. Mycobacterium tuberculosis complex (MTBC) genetic heterogeneity, and differential host immunological responses, have been implicated in its pathogenesis. METHODS: To determine the association between MTBC lineage and PTB disease phenotype, we used two retrospective cohorts of PTB patients in Canada and two independent lineage attribution methods (DNA fingerprinting and genome sequencing). The first cohort, Cohort 1, consisted of consecutively diagnosed PTB patients between 2014 and 2020. The second, Cohort 2, consisted of newly-arrived foreign-born PTB patients who either were or were not referred for post-landing medical surveillance between 2004 and 2017. Univariable and multivariable logistic regression models were sequentially fitted to both cohorts, adjusting for age, sex, disease type, drug resistance and HIV. Evolution of radiographic features was correlated to lineage in Cohort 2. FINDINGS: Cohort 1 and 2 included 874 (209 subclinical) and 111 (44 subclinical) patients, respectively. In both cohorts, subclinical patients were more likely than clinical patients to have relapse/retreatment disease, be smear-negative, have longer times-to-culture positivity and to harbor an ancestral MTBC lineage (Indo-Oceanic or Mycobacterium africanum). Relapse/retreatment disease and ancestral MTBC lineage were independent predictors of subclinical disease (ORs and 95% CIs in Cohort 1, 1.85 [1.07,3.28], p < 0.029 and 2.30 [1.66,3.18], p < 0.001, respectively, and Cohort 2, 5.74 [1.37-24.06], p < 0.017 and 3.21 (1.29,7.97], p < 0.012, respectively). The geographic distribution of Indo-Oceanic strains causing subclinical disease was uneven. Non-progressive lung disease was more common in patients infected with ancestral than modern lineages in Cohort 2, 56.0% vs 25.4%, p < 0.005. INTERPRETATION: MTBC lineage is a strong predictor of PTB disease phenotype. The genetic drivers of this association, and the relative contribution of other explanatory variables, are unknown.

Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population.

Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent agreement was 99%. GPP, a multi-analyte platform can easily be implemented into a routine diagnostic laboratory for detecting enteric pathogens including C. difficile.

Molecular Detection of Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) Directly from Stool Using Multiplex qPCR Assays.

Non-O157 Shiga toxin-producing E. coli (STEC) can cause outbreaks that have great economic and health impact. Since the implementation of STEC screening in Alberta in 2018, it is also essential to have a molecular serotyping method with faster turnaround time for cluster identification and surveillance purposes. This study sought to perform molecular serotyping of the top six non-O157 (O26, O45, O103, O111, O121 and O145) STEC serotypes directly from stools and enrichment broths compared to conventional methods on isolates. Multiplex, serotyping qPCR assays were used to determine sensitivity and specificity of the top six non-O157 STEC serotypes. Sensitivity and specificity were assessed for both singleplex and multiplex qPCR assays for comparison of the top six serotypes. Blinded stool specimens (n = 116) or broth samples (n = 482) submitted from frontline microbiology laboratories for STEC investigation were analyzed by qPCR. Both singleplex and multiplex assays were comparable, and we observed 100% specificity with a limit of detection of 100 colony-forming units per mL. Direct molecular serotyping from stool specimens mostly correlated (88%) with conventional serotyping of the cultured isolate. In cases of discordant serotypes, the top six non-O157 STEC mixed infections were identified and confirmed by culture and conventional serotyping. Detection of non-O157 STEC can be done directly from stool specimens using multiplex PCR assays with the ability to identify mixed infections, which would otherwise remain undetected by conventional serotyping of a single colony. This method can be easily implemented into a frontline diagnostic laboratory to enhance surveillance of non-O157 STEC, as more frontline microbiology laboratories move to culture independent assays.

Comparison of Common Enrichment Broths Used in Diagnostic Laboratories for Shiga Toxin-Producing Escherichia coli.

Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture-independent diagnostic testing; however, recovery of STEC remains an important aspect of surveillance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep's blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the optimal enrichment for growth of isolates with and without stool. The results from this study suggest that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment instead of GN or Mac.

The effect of a microbial ecosystem therapeutic (MET-2) on recurrent Clostridioides difficile infection: a phase 1, open-label, single-group trial.

BACKGROUND: Faecal microbiota transplantation (FMT) is highly effective for recurrent Clostridioides difficile infection but has inherent risks. Microbial Ecosystem Therapeutic 2 (MET-2) is an oral encapsulated formulation of 40 lyophilised bacterial species initially isolated from stool of a healthy donor, but subsequently manufactured independently of donors, eliminating potential risks introduced by changes in donor health. The aim of this study was to determine MET-2 activity, safety, and tolerability. METHODS: This phase 1, open-label, single-group feasibility study was done in Alberta, Canada. The main inclusion criteria were mild to moderate C difficile infection and at least one episode of C difficile infection recurrence (ie, two episodes of C difficile infection) within 12 months. Initial daily treatment was ten oral capsules for 2 days, then three capsules for 8 days. If C difficile infection recurred, a higher dose was offered: 20 capsules for 2 days, then three capsules for 8 days. Patients were followed for adverse events and C difficile infection recurrence up to day 130. The primary outcome was absence of C difficile infection recurrence (fewer than three unformed bowel movements in 24 h persisting for at least 2 days) at day 40 by intention-to-treat analysis. Secondary outcomes were mortality or hospitalisation due to C difficile infection, infections attributed to treatment, nausea, abdominal pain, vomiting, or diarrhoea during treatment, quality of life (C difficile Health Related Quality of Life Questionnaire) before and after treatment, and engrafted MET-2 bacteria in patient stool. Absence of C difficile infection recurrence at day 130 was an exploratory outcome. This study is registered with ClinicalTrials.gov, NCT02865616 FINDINGS: Between Sept 19, 2018, and Feb 28, 2020, we enrolled 19 adult patients with at least two episodes of mild to moderate C difficile infection (median age 65 years [IQR 56-67]; 12 women [63%], seven men [37%]). Recurrent C difficile infection was absent at day 40 in 15 (79%) of 19 patients after initial treatment, increasing to 18 (95%) 40 days after retreatment. No mortality associated with C difficile infection, infections associated with MET-2 treatment, or other serious adverse events were observed. The most common self-limited, mild to moderate symptoms reported during treatment were diarrhoea in 12 (63%) of 19 patients and abdominal cramps in 12 (63%). After MET-2 treatment, quality of life improved significantly, as did alpha diversity in stool microbial composition (p=1·93×10-6). MET-2 associated taxa were found in greater abundance in most patients after treatment compared with baseline. 16 (84%) of 19 patients did not have recurrence of C difficile infection by day 130. INTERPRETATION: MET-2 appears to be safe, efficacious, and well tolerated among patients with recurrent C difficile infection. Results must be validated in controlled studies. FUNDING: NuBiyota.

Direct Clostridioides difficile ribotyping from stool using capillary electrophoresis.

Clostridioides difficile(C. difficile) genotyping is essential for surveillance of emerging strains, transmissions, and outbreak investigations, but culture is lengthy and may not be routinely performed, which necessitates culture-independent genotyping methods. We aimed to develop a direct from stool C. difficile PCR ribotyping algorithm using capillary electrophoresis. Ribotypes were generated directly from 66.8% of stools with 33.2% requiring broth enrichment. 16S and tcdB cycle thresholds (Ct) were significantly lower (P< 0.001) in directly ribotyped stools compared to enriched stools, and Ct correlated with direct ribotyping (area under the curve: 0.97 and 0.96, respectively). Direct and isolate ribotypes were 94.7% concordant. Mixed C. difficile ribotypes were presumptively identified in 14 (7.5%) samples with 12 (6.4%) mixtures confirmed. We have developed a rapid PCR ribotyping algorithm allowing for direct C. difficile genotyping from stool using capillary electrophoresis with occasional detection of mixed C. difficile populations in stool, which is a limitation of conventional isolate genotyping.

Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC).

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes' amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3-5 days until a sample is negative by culture.

A randomized trial evaluating virus-specific effects of a combination probiotic in children with acute gastroenteritis.

Gastroenteritis accounts for nearly 500,000 deaths in children younger than 5 years annually. Although probiotics have been touted as having the potential to expedite diarrhea resolution, recent clinical trials question their effectiveness. A potential explanation is a shift in pathogens following the introduction of a rotavirus vaccine. Here, we report the results of a multi-center, double-blind trial of 816 children with acute gastroenteritis who completed follow-up and provided multiple stool specimens. Participants were randomized to receive a probiotic containing Lactobacillus rhamnosus and Lactobacillus helveticus or placebo. We report no virus-specific beneficial effects attributable to the probiotic, either in reducing clinical symptoms or viral nucleic acid clearance from stool specimens collected up to 28 days following enrollment. We provide pathophysiological and microbiologic evidence to support the clinical findings and conclude that our data do not support routine probiotic administration to children with acute gastroenteritis, regardless of the infecting virus.

The physiotherapy management of patients undergoing coronary artery surgery: A questionnaire survey.

The current physiotherapy management of patients undergoing coronary artery surgery in 22 public and 13 private hospitals across Australia and New Zealand was examined using a questionnaire survey. Respondents were asked to identify assessment and treatment techniques used in the pre- and post-operative management. An 83 per cent response from physiotherapists was obtained. Ninety-four per cent of respondents reported that pre-operative assessment was performed routinely. Eighty-nine per cent of respondents indicated that all patients were treated routinely by physiotherapists in the post-extubation period. Positioning and deep breathing exercises were the most commonly used techniques for patients post-extubation. Factors determining treatment choice and the impact of research on current physiotherapy practice are discussed.