Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Trends in Clostridioides difficile infection rates in Canadian hospitals during the coronavirus disease 2019 (COVID-19) pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has placed significant burden on healthcare systems. We compared Clostridioides difficile infection (CDI) epidemiology before and during the pandemic across 71 hospitals participating in the Canadian Nosocomial Infection Surveillance Program. Using an interrupted time series analysis, we showed that CDI rates significantly increased during the COVID-19 pandemic.

Characterization of Healthcare-Associated and Community-Associated Clostridioides difficile Infections among Adults, Canada, 2015-2019.

We investigated epidemiologic and molecular characteristics of healthcare-associated (HA) and community-associated (CA) Clostridioides difficile infection (CDI) among adult patients in Canadian Nosocomial Infection Surveillance Program hospitals during 2015-2019. The study encompassed 18,455 CDI cases, 13,735 (74.4%) HA and 4,720 (25.6%) CA. During 2015-2019, HA CDI rates decreased by 23.8%, whereas CA decreased by 18.8%. HA CDI was significantly associated with increased 30-day all-cause mortality as compared with CA CDI (p<0.01). Of 2,506 isolates analyzed, the most common ribotypes (RTs) were RT027, RT106, RT014, and RT020. RT027 was more often associated with CDI-attributable death than was non-RT027, regardless of acquisition type. Overall resistance C. difficile rates were similar for all drugs tested except moxifloxacin. Adult HA and CA CDI rates have declined, coinciding with changes in prevalence of RT027 and RT106. Infection prevention and control and continued national surveillance are integral to clarifying CDI epidemiology, investigation, and control.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients.

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab. A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%. Average viral target Ct values were higher in the saline gargle samples. RNaseP Ct values were lower in unobserved collected samples compared to observed collected samples. Unobserved self-collection of saline gargle samples is a promising outpatient testing method for COVID-19 diagnosis. The self-collection method has potential to simplify the diagnostic cycle and facilitate implementation of COVID-19 testing, particularly in settings with limited access to health care workers.

COVID-19 in an immunocompromised host: persistent shedding of viable SARS-CoV-2 and emergence of multiple mutations: a case report.

This article reports a case of a 21-year-old woman with refractory B-cell acute lymphocytic leukaemia who presented with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). She remained positive for SARS-CoV-2 by viral culture for 78 days and by polymerase chain reaction (PCR) for 97 days. Sequencing of repeat samples over time demonstrated an increasing and dynamic repertoire of mutations.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients.

We assessed the performance, stability, and user acceptability of swab-independent self-collected saliva and saline mouth rinse/gargle sample types for the molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adults and school-aged children. Outpatients who had recently been diagnosed with COVID-19 or were presenting with suspected COVID-19 were asked to have a nasopharyngeal (NP) swab collected and provide at least one self-collected sample type. Participants were also asked about sample acceptability using a five-point Likert scale. For those previously diagnosed with COVID-19, all samples underwent real-time PCR testing using a lab-developed assay, and the majority were also tested using an FDA-authorized assay. For those presenting with suspected COVID-19, only those with a positive nasopharyngeal swab sample went on to have other samples tested. Saline mouth rinse/gargle and saliva samples were tested daily at time zero, day 1, and day 2 to assess nucleic acid stability at room temperature. Fifty participants (aged 4 to 71 years) were included; of these, 40 had at least one positive sample and were included in the primary sample yield analysis. Saline mouth rinse/gargle samples had a sensitivity of 98% (39/40), while saliva samples had a sensitivity of 79% (26/33). Both saline mouth rinse/gargle and saliva samples showed stable viral RNA detection after 2 days of room temperature storage. Mouth rinse/gargle samples had the highest (mean, 4.9) and health care worker (HCW)-collected NP swabs had the lowest acceptability scores (mean, 3.1). In conclusion, saline mouth rinse/gargle samples demonstrated higher combined user acceptability ratings and analytical performance than saliva and HCW-collected NP swabs. This sample type is a promising swab-independent option, particularly for outpatient self-collection in adults and school-aged children.

Septic arthritis due to Mycoplasma orale in a young patient with hypogammaglobulinemia.

Mycoplasma orale is an obligate intracellular bacterium usually found as a commensal in the human oral cavity. Symptomatic infections with this organism are rare, but severe disease has been described in the setting of impaired humoral immunity. Here, we describe a case in which M. orale was identified from the joint fluid of a patient with septic arthritis, splenic lesions, and agammaglobulinemia. A 15-year-old boy was admitted to the hospital with fever, progressive left knee swelling, and pain. His medical history was significant for Burkitt's lymphoma, the treatment of which had included rituximab 6 years earlier. M. orale was identified in the synovial fluid using 16S ribosomal RNA gene sequencing. He was also found to be hypogammaglobulinemic, and imaging revealed multiple splenic lesions. He was treated with doxycycline and intravenous immunoglobulin, which resulted in complete resolution of his arthritis and other symptoms. Mycoplasma species should be suspected in patients with humoral immunodeficiency and compatible findings.

Le Mycoplasma orale est une bactérie intracellulaire obligatoire généralement observée dans la flore commensale de la cavité orale. Les infections symptomatiques par ces organismes sont rares, mais une maladie grave a été décrite en cas de perturbation de l'immunité humorale. Dans le présent document, les auteurs décrivent un cas de M orale décelé dans le liquide articulaire d'un patient atteint d'arthrite septique, de lésions spléniques et d'agammaglobulinémie. Un garçon de 15 ans a été hospitalisé parce qu'il faisait de la fièvre, avait un œdème évolutif du genou gauche et de la douleur. Son histoire médicale incluait un lymphome de Burkitt, dont le traitement comprenait du rituximab six ans plus tôt. Le M orale a été décelé dans le liquide synovial au moyen du séquençage du gène d'ARN ribosomique 16S. Il était également hypogammaglobulinémique, et l'imagerie a révélé de multiples lésions spléniques. Il a reçu un traitement à la doxycycline et aux immunoglobulines intraveineuses, qui ont favorisé une résolution complète de son arthrite et de ses autres symptômes. Il faut envisager des espèces de Mycoplasma chez les patients ayant une immunodéficience humorale et des observations compatibles.

Contribution of Real-Time Xpert MTB/RIF Testing to Establishing Early Diagnosis of Pediatric Extrapulmonary Tuberculosis in a Nonendemic Setting: A Case Series.

Microbiologic confirmation of pediatric extrapulmonary tuberculosis remains challenging, leading to diagnostic delays. In our retrospective case series, real-time molecular testing (Xpert MTB/RIF) on respiratory and extrapulmonary specimens resulted in a more rapid diagnosis of extrapulmonary tuberculosis in a nonendemic, high resource setting.

Asymptomatic Pharyngeal Carriage of Kingella kingae Among Young Children in Vancouver, British Columbia, Canada.

BACKGROUND: Kingella kingae has emerged as a significant cause of osteoarticular infections in young children. Pharyngeal colonization is considered a prerequisite for invasive K. kingae infection. We conducted a prospective study to estimate the prevalence of pharyngeal carriage of K. kingae among healthy young children in Vancouver. METHODS: From March 2016 to May 2017, children between 6 and 48 months of age visiting British Columbia Children's Hospital outpatient clinics for noninfectious causes were included in the study. Another set of participants was enrolled from a day-care center located at British Columbia Children's Hospital. A single-throat swab was collected after obtaining consent from parent/guardian. The samples were stored at -70°C and tested using an in-house developed real-time polymerase chain reaction assay. Epidemiologic characteristics and risk factors for K. kingae colonization were collected via a study questionnaire. RESULTS: A total of 179 children were enrolled in the study, but only 174 samples were eligible for testing. Of the 174 samples, 5 had indeterminate results and the remaining 169 samples were negative by K. kingae polymerase chain reaction. The median age of participants was 23 months. About 36% of children were attending day care and had another sibling <5 years of age. Previous history of cold symptoms and antibiotic use was reported in 42% and 12%, respectively. CONCLUSIONS: The results of our study showed no prevalence of asymptomatic pharyngeal carriage of K. kingae in young children in Vancouver. Additional multicenter studies may help to understand the differences in pharyngeal carriage rate among healthy children.

Prevalence and clinical impact of methicillin-resistant Staphylococcus aureus colonization among infants at a level III neonatal intensive care unit.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known nosocomial pathogen in neonatal intensive care unit (NICU) patients. Studies on the impact of MRSA colonization on neonatal morbidities are scarce. METHODS: We conducted a 1:3 matched cohort study among infants with and without MRSA colonization, born between January 2010 and June 2014, in a tertiary NICU to review their demographic characteristics and outcomes. RESULTS: During the study period, rates of MRSA colonization and bacteremia were found to be 0.68% and 0.10%, respectively. No differences in demographic characteristics, mortality, and major morbidities were identified among infants with and without MRSA colonization. CONCLUSIONS: We reported a low rate of MRSA colonization in infants admitted to our NICU, without impact on mortality and inhospital morbidity. Further large-scale studies are needed to understand the implications and cost-effectiveness of active MRSA surveillance.

Baylisascariasis: A young boy with neural larva migrans due to the emerging raccoon round worm.

A 17-month-old boy from Vancouver, Canada, presented with a 5-day history of progressive somnolence, ataxia, and torticollis. Additional investigations revealed eosinophilic encephalitis with deep white matter changes on MR imaging. On day 13, serology came back positive for Baylisascaris procyonis antibodies. While prophylaxis after ingestion of soil or materials potentially contaminated with raccoon feces can prevent baylisascariasis, timely treatment can sometimes alter a disastrous outcome. Populations of infected raccoons are propagating globally, but cases of Baylisascaris neural larva migrans have so far only been reported from North America.

Pediatric oncology and stem cell transplant patients with healthcare-associated Clostridium difficile infection were already colonized on admission.

Clostridium difficile is the leading cause of healthcare-associated infections worldwide. The diagnosis of C. difficile infection (CDI) in pediatric oncology patients is complex as diarrhea is common, and there is a high rate of colonization in infants and young children. This study was conducted to assess the accuracy of the surveillance definitions of healthcare-associated CDI (HA-CDI) and to determine the prevalence of toxigenic C. difficile colonization among pediatric oncology and stem cell transplant patients. METHODS: A prospective cohort study was conducted over a three-year period in an inpatient pediatric oncology and stem cell transplant setting. Baseline stool samples were collected within three days of admission and were genotypically compared with clinically indicated samples submitted after three days of admission. RESULTS: A total of 175 patients were recruited with a total of 536 admissions. The adjusted prevalence of baseline toxigenic C. difficile colonization among admissions was 32.8%. Seventy-eight percent of positive admissions did not have history of CDI. Colonization with a toxigenic strain on admission was predictive of CDI (OR = 28.6; 95% CI, 6.58-124.39; P < 0.001). Nearly all clinical isolates (8/9) shared identical pulsed-field gel electrophoresis patterns with baseline isolates or were closely related (1/9). Only one of the 11 cases that were considered HA-CDI was potentially nosocomially acquired. CONCLUSION: The prevalence of colonization with toxigenic C. difficile in our cohort is high. Unfortunately, the current CDI surveillance definitions overestimate the incidence of HA-CDI in pediatric oncology and stem cell transplantation settings.

Measles Lymphadenopathy in a Child With PFAPA Syndrome.

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is a common cause of periodic fever in children. The pathogenesis of PFAPA is unknown but likely involves immune system dysregulation and may be initiated by an environmental trigger. Tonsillectomy resolves or improves symptoms in some patients, but the reason for this is unknown; moreover, specific abnormalities in tonsillectomy specimens from PFAPA patients have not been described. Here, we report measles virus in tonsil from a child with PFAPA. Measles-type viral cytopathic effect was discovered on histological examination of tonsillar tissue after therapeutic tonsillectomy for PFAPA. Molecular testing showed the left tonsil was positive for measles RNA by reverse transcription polymerase chain reaction (RT-PCR) while the right tonsil was inconclusive (weakly positive). Real-time RT-PCR specific for measles vaccine strain RNA (genotype A) was weakly reactive in the left tonsil tissue when tested in 3 independent replicates, but this result could not be confirmed with conventional genotyping by sequencing. The relationship and clinical significance between measles virus and PFAPA in this case is unclear but may be related to PFAPA-associated immune dysregulation. Additional investigation of measles virus in PFAPA patients would be helpful in further exploring this potential association.

Evaluation of amplification targets for the specific detection of Bordetella pertussis using real-time polymerase chain reaction.

BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity. METHODS: A novel B pertussis real-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481, ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including different Bordetella species and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products. RESULTS: Analytical sensitivity was highest for the assay targeting the IS481 element; however, the assay lacked specificity for B pertussis in the reference panel and in the clinical samples. False-positive results were also observed with assays targeting the ptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains of B pertussis. However, a novel assay targeting the porin gene demonstrated high specificity for B pertussis both in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted all B pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction. CONCLUSION: A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.

HISTORIQUE: Les infections à Bordetella pertussis continuent d'être un important problème de santé publique au Canada. Les méthodes de réaction en chaîne de la polymérase (PCR) pour déceler le B pertussis sont habituellement fondées sur la séquence d'insertion multicopie IS481, dont la sensibilité élevée, mais dont la spécificité d'espèce est inexistante. MÉTHODOLOGIE: Une nouvelle méthode PCR en temps réel du B pertussis fondée sur le gène de porine a été mise à l'essai parallèlement à plusieurs méthodes déjà publiées qui ciblent des gènes comme l'IS481, le promoteur de ptx, la pertactine et une thialase éventuelle. Les méthodes ont été évaluées à l'aide d'un groupe de référence de bactéries respiratoires communes, y compris diverses espèces de Bordetella et 107 échantillons nasopharyngés cliniques. Les résultats contradictoires ont été confirmés par séquençage des produits de PCR. RÉSULTATS: La méthode visant l'élément IS481 avait la sensibilité analytique la plus élevée, mais manquait de spécificité pour le B pertussis dans le groupe de référence et les échantillons cliniques. Les méthodes ciblant les gènes du promoteur de ptx et de la perctatine ont également donné des résultats faux positifs. Une méthode de PCR fondée sur le gène thialase était hautement spécifique, mais ne décelait pas toutes les souches de référence du B pertussis. Cependant, une nouvelle méthode ciblant le gène de porine a démontré une forte spécificité au B pertussis, à la fois dans le groupe de référence et dans les échantillons cliniques et, d'après les résultats confirmés par séquençage, prédit correctement tous les cas positifs au B pertussis dans les échantillons cliniques. D'après l'analyse de régression Probit, la limite de détection de 95 % de la nouvelle méthode était de quatre unités formant colonies par réaction. CONCLUSION: Une nouvelle méthode faisant appel à la porine pour déceler le B pertussis donne un rendement supérieur et peut être utile pour améliorer la détection moléculaire du B pertussis dans des échantillons cliniques.

Granulomatous herpes simplex encephalitis in an infant with multicystic encephalopathy: a distinct clinicopathologic entity?

BACKGROUND: Herpes simplex virus encephalitis can manifest as a range of clinical presentations including classic adult, neonatal, and biphasic chronic-granulomatous herpes encephalitis. METHOD: We report an infant with granulomatous herpes simplex virus type 2 encephalitis with a subacute course and multicystic encephalopathy. CASE: A 2-month-old girl presented with lethargy and hypothermia. Computed tomography scan of the head showed multicystic encephalopathy and calcifications. Cerebrospinal fluid analysis by polymerase chain reaction testing for herpes simplex virus 1 and 2, enterovirus, and cytomegalovirus was negative. Normal cerebrospinal fluid interferon-α levels argued against Aicardi-Goutières syndrome. The patient died 2 weeks after presentation. At autopsy, multicystic encephalopathy was confirmed with bilateral gliosis, granulomatous inflammation with multinucleated giant cells, and calcifications. Bilateral healing necrotizing retinitis suggested a viral etiology, but retina and brain were free of viral inclusions and immunohistochemically negative for herpes simplex virus-2 and cytomegalovirus. However, polymerase chain reaction analysis showed herpes simplex virus-2 DNA in four cerebral paraffin blocks. Subsequent repeat testing of the initial cerebrospinal fluid sample using a different polymerase chain reaction assay was weakly positive for herpes simplex virus-2 DNA. CONCLUSION: Granulomatous herpes simplex virus encephalitis in infants can present with subacute course and result in multicystic encephalopathy with mineralization and minimal cerebrospinal fluid herpes simplex virus DNA load. Infectious etiologies should be carefully investigated in the differential diagnosis of multicystic encephalopathy with mineralization, in particular if multinucleated giant cells are present.