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.

Surveillance of mosquito-borne viruses in Alberta using reverse transcription polymerase chain reaction with generic primers.

Mosquitoes collected during 2003, 2004, and 2005 in Alberta, Canada, were screened for the presence of a wide range of arboviruses by reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acid extracts from mosquito slurries were amplified using universal primers designed to detect viruses belonging to the Flavivirus genus of the Flaviviridae family and California and Bunyamwera serogroups of the Bunyavirus genus within the Bunyaviridae family. Species-specific detection of Western equine encephalitis virus and Eastern equine encephalitis virus was also performed. Amplified products were analyzed, and the viral target was identified by sequencing. Of the 418 pools tested, 3 pools contained Cache Valley virus belonging to Bunyaviridae and 103 pools were positive for a previously undescribed flaviviral sequence that was most similar to Kamiti River virus. These data suggest that nucleic acid amplification using broadly reactive primers can be adopted for arbovirus surveillance in mosquito populations, and this approach has the potential to detect both previously recognized and novel viruses.

Screening of organ and tissue donors for West Nile virus by nucleic acid amplification--a three year experience in Alberta.

West Nile Virus (WNV)-specific nucleic acid amplification testing (NAAT) of organ and tissue donors remains controversial. We report three years of WNV donor screening in Alberta Canada using NAAT. Between 2003 and 2005, 1549 initial specimens were received. A valid negative result was issued within the specified turnaround time on 1531 (98.8%). The initial NAAT was successful for 1393 samples (90%), while repeat testing using an alternate NAAT resolved a further 126 samples. For 12 of 14 donors, a second specimen provided a valid negative result. Failure to generate a valid negative result in time resulted in rescheduling of one living related organ transplant, and surgery proceeded in the absence of a final result in one multi-organ donation after risk assessment. For 11 tissue donors, tissues were discarded due to lack of a WNV result. Invalid results usually occurred on postmortem haemolyzed tissue donor samples due to inhibitory reactions. There were no confirmed positive donors, no false-positive results and no solid organs lost due to WNV testing. We conclude that WNV NAAT of organ and tissue donors can be implemented without compromising availability of donors but requires committed laboratory support.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes.

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

Maculopapular rash and tremor are associated with West Nile fever and neurological syndromes.

The clinical features associated with West Nile virus (WNV) infections are described based on data collected from history forms submitted with samples during a province-wide WNV testing programme. Age 40-59 years (OR 1.7, p<0.008), residence in the southeast of Alberta (OR 4.2, p<0.001), maculopapular rash (OR 8.6, p<0.001) or tremor (OR 3.6, p<0.001) were independently associated with WNV infection.

Impact of Matrix-Assisted Laser Desorption and Ionization Time-of-Flight and Antimicrobial Stewardship Intervention on Treatment of Bloodstream Infections in Hospitalized Children.

BACKGROUND.: Early definitive identification of infectious pathogens coupled with antimicrobial stewardship interventions allow for targeted and timely administration of antimicrobials. We investigated the combined impact of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology and an antimicrobial stewardship program (ASP) in pediatric patients with blood stream infections (BSIs). METHODS.: This is a single-center study comparing a control group of patients from October 2009 to July 2010 with BSIs to a cohort of patients postimplementation of MALDI-TOF and an ASP, from October 2013 to July 2014. Primary outcome was time to optimal therapy. Secondary outcomes included time to effective therapy, 30-day all-cause mortality, 30-day readmission rate, hospital length of stay, and intensive care admission. RESULTS.: One hundred episodes of BSIs were identified in the preintervention period, and 121 episodes were identified in the postintervention period. Time from blood culture collection to organism identification was significantly reduced in the prospective cohort compared with historical controls (18.8 vs 43.7 hours, respectively). A total of 73 ASP interventions were made on the treatment of BSIs in the postintervention period. Combined use of MALDI-TOF and ASP significantly reduced time to optimal therapy (77.0 to 54.2 hours, P < .001). In the subgroup analysis of Gram-negative bacteremia, time to effective and optimal therapy were significantly reduced (2.0 vs 0.7 hours and 146.8 vs 48.0 hours, respectively). There were no significant differences in clinical outcomes. CONCLUSIONS.: The combined use of MALDI-TOF and ASP allows early optimization of antimicrobial therapy in pediatric inpatients with BSIs.

Use of sequence-based typing for investigation of a case of nosocomial legionellosis.

A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.

Evaluation of real-time PCR for diagnosis of Bordetella pertussis infection.

BACKGROUND: Nucleic acid amplification of the IS481 region by PCR is more sensitive than culture for detection and diagnosis of Bordetella pertussis but the assay has known cross-reactivity for Bordetella holmesii and its use as a routine diagnostic assay has not been widely evaluated. METHODS: The objectives of this study were: 1) to assess the diagnostic utility of real-time IS481 PCR by comparison of results with culture and direct fluorescent antigen (DFA) testing for B. pertussis, 2) to employ a PCR assay designed against a different insertion sequence (IS1001) to assess the incidence of B. holmesii in symptomatic individuals and 3) to design and evaluate a new PCR-based assay which could be used for B. pertussis confirmation. A total of 808 nasopharyngeal specimens were included in the study the majority of which were submitted in charcoal transport medium (88%) with the rest submitted in Regan-Lowe medium. RESULTS: Concordant results for PCR, DFA and culture were obtained for 21 B. pertussis positive and 729 B. pertussis negative specimens. DFA was prone to false positive and negative reactions when compared with both PCR and culture. The IS481 PCR identified 28 positive results for specimens that were DFA and culture negative. A novel real-time PCR targeting the B. pertussis toxin promoter was found to be specific and useful for confirming the majority of IS481 positive specimens as B. pertussis. B. holmesii was not detected in any of the submitted samples. CONCLUSION: The potential pick up of B. holmesii by the IS481 PCR had minimal diagnostic relevance in the Alberta population during the time period of our study. The IS481 PCR assay is now used in our laboratory routinely for front-line screening of samples for B. pertussis with associated enhancement in diagnostic sensitivity compared with DFA and culture. Retrospectively, patients' samples are batched and tested by the IS1001 MB and TPR assays for research purposes and to ensure there is no change in B. holmesii incidence in the population.

Comparison between the clinical and laboratory features of enterovirus and West Nile virus infections.

The seasonality and clinical features of enterovirus (EV) infections overlap with those of West Nile virus (WNV). The purpose of this study was to determine the frequency of EV detection in patients being tested for WNV and to look for features that could be used to distinguish between infections with these two viruses. Nucleic acid amplification testing (NAT) for EV was performed on all plasma samples submitted for WNV testing in 2003 and 2004. Demographics, clinical features, and laboratory results for patients with documented EV viremia were compared with those for patients with confirmed WNV infection (as diagnosed by NAT and/or serology). NAT for EV was positive on 50 of 1,784 serum or plasma samples submitted for WNV testing (2.8%). Clinical information was compared for 45 patients with EV viremia and 214 patients with WNV infection. Patients with EV viremia were younger and less likely to have heart disease or a travel history (P<0.05). The EV viremia cases were distributed throughout the whole province while the WNV cases were predominantly in the southern part of the province. Symptoms were remarkably similar, although patients with WNV infection were more likely to have anorexia, dizziness, rash, and cranial nerve palsy (P<0.05). There are no consistent differences in the features of WNV infection and enteroviral viremia so diagnostic tests for both viruses should be performed when WNV is present in local mosquitoes.

Microfluidic device for dielectrophoresis manipulation and electrodisruption of respiratory pathogen Bordetella pertussis.

A miniaturized microfluidic device was developed to facilitate electromanipulation of bacterial respiratory pathogens. The device comprises a microchip with circular aluminum electrodes patterned on glass, which is housed in a microfluidic system fabricated utilizing polydimethylsiloxane. The device provides sample preparation capability by exploiting positive dielectrophoresis (DEP) in conjunction with pulsed voltage for manipulation and disruption of Bordetella pertussis bacterial cells. Positive DEP capture of B. pertussis was successfully demonstrated utilizing 10 Vrms and 1 MHz ac fields. Application of dc pulses (300 V amplitude and 50 micros pulsewidth applied 1 s apart) across the aluminum electrodes resulted in electrodisruption and lysis of B. pertussis bacterial cells. Real-time polymerase chain reaction, a 2(3) factorial experimental design and transmission electron microscopy were used to evaluate bacterial cell manipulation and factors affecting bacterial cell disruption. The main factors affecting bacterial cell disruption were electric field strength, the electrical conductivity of the cell suspension sample, and the combined effect of number of pulses and sample conductivity. The bacterial deoxyribonucleic acid target remained undamaged as a result of DEP and cell lysis experimentation. Our findings suggest that a simple miniaturized microfluidic device can achieve important steps in sample preparation on-chip involving respiratory bacterial pathogens.

Integrated microfluidic systems for sample preparation and detection of respiratory pathogen Bordetella pertussis.

An integrated microfluidic system for combined manipulation, pre-concentration, and lysis of samples containing Bordetella pertussis by dielectrophoresis and electroporation has been developed and implemented. The microfluidic device was able to pre-concentrate the amount of B. pertussis cells present in 200 microl of a B. pertussis suspension stock into a 20 microl volume. The device exhibited optimal sample pre-concentration of 6.7x at a stock value of 10(3) cfu/ml and at a flow rate of 250 microl/h. Electro-disruption experiments showed that on-chip-based electroporation is an effective solution for lysis of B. pertussis cells that is easily integrated with dielectrophoresis assisted pre-concentration procedures. Pulsed voltage applied, number of pulses, and presence of potassium chloride in a B. pertussis suspension showed a reduction in B. pertussis cell viability by electroporation; and transmission electron microscopy confirmed B. pertussis cell disruption by electroporation. Genetic amplification and detection of the pre-concentrated sample employing an integrated chip-based system demonstrated a complete chip approach for pathogen detection.

Nucleic acid testing for west nile virus RNA in plasma enhances rapid diagnosis of acute infection in symptomatic patients.

Although nucleic acid amplification testing (NAAT) for West Nile virus (WNV) is useful in screening blood donors, such methods have not been studied in symptomatic patients. For diagnosis of WNV infection, 1.0 mL of plasma was tested by NAAT, and WNV-specific immunoglobulin M was assayed. Of 276 WNV cases, 191 were tested by both serology and NAAT. Of these, 86 (45.0%), 111 (58.1%), and 180 (94.2%) were detected by NAAT, serology, and combined NAAT and serology, respectively. NAAT-based screening was most useful within 8 days of the onset of symptoms. Viremia is common in early symptomatic WNV infection, and NAAT enhances diagnostic yield.

Clinical utility of commercial enzyme immunoassays during the inaugural season of West Nile virus activity, Alberta, Canada.

West Nile virus (WNV) has spread rapidly across North America, creating a need for rapid and accurate laboratory diagnosis on a large scale. Immunoglobulin M (IgM) capture enzyme immunoassays (EIA) became commercially available in the summer of 2003, but limited data are available on their clinical performance. Consolidated human WNV diagnostic testing for the province of Alberta, Canada, at the public health laboratory permitted a large-scale evaluation of the assays, covering a wide clinical spectrum. Two thousand nine hundred sixty-nine sera were tested, from 2,553 Alberta residents, and 266 cases were identified. Sensitivities of the Focus assay and first-generation Panbio IgM capture EIA were 79 and 80%, respectively. During the first week of illness only 53 to 58% of cases were positive, but sensitivity was 96 to 97% after day 8. Sensitivity for neurological cases was 92% overall. Specificity was high for the Focus kit at 98.9%, but only 82.9% for the first Panbio kit. A positive Focus WNV IgG result with a twofold rise in IgG index was a reliable indicator of acute flavivirus infection (67/67 WNV). Agreement between the IgG test and hemagglutinin inhibition titers in paired sera was at least 82%. Commercial IgM and IgG EIA proved useful for WNV diagnosis, provided follow-up sera were collected after 8 days of illness.