The role of the microbiota in respiratory virus-bacterial pathobiont relationships in the upper respiratory tract.

The mechanisms by which respiratory viruses predispose to secondary bacterial infections remain poorly characterized. Using 2,409 nasopharyngeal swabs from 300 infants in Botswana, we performed a detailed analysis of factors that influence the dynamics of bacterial pathobiont colonization during infancy. We quantify the extent to which viruses increase the acquisition of Haemophilus influenzae , Moraxella catarrhalis , and Streptococcus pneumoniae . We provide evidence of cooperative interactions between these pathobionts while identifying host characteristics and environmental exposures that influence the odds of pathobiont colonization during early life. Using 16S rRNA gene sequencing, we demonstrate that respiratory viruses result in losses of putatively beneficial Corynebacterium and Streptococcus species that are associated with a lower odds of pathobiont acquisition. These findings provide novel insights into viral-bacterial relationships in the URT of direct relevance to respiratory infections and suggest that the URT bacterial microbiota is a potentially modifiable mechanism by which viruses promote bacterial respiratory infections.

SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

SARS-CoV-2 Outbreak Investigation Using Contact Tracing and Whole-Genome Sequencing in an Ontario Tertiary Care Hospital.

Genomic epidemiology can facilitate an understanding of evolutionary history and transmission dynamics of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak. We used next-generation sequencing techniques to study SARS-CoV-2 genomes isolated from patients and health care workers (HCWs) across five wards of a Canadian hospital with an ongoing SARS-CoV-2 outbreak. Using traditional contact tracing methods, we show transmission events between patients and HCWs, which were also supported by the SARS-CoV-2 lineage assignments. The outbreak predominantly involved SARS-CoV-2 B.1.564.1 across all five wards, but we also show evidence of community introductions of lineages B.1, B.1.1.32, and B.1.231, falsely assumed to be outbreak related. Altogether, our study exemplifies the value of using contact tracing in combination with genomic epidemiology to understand the transmission dynamics and genetic underpinnings of a SARS-CoV-2 outbreak. IMPORTANCE Our manuscript describes a SARS-CoV-2 outbreak investigation in an Ontario tertiary care hospital. We use traditional contract tracing paired with whole-genome sequencing to facilitate an understanding of the evolutionary history and transmission dynamics of this SARS-CoV-2 outbreak in a clinical setting. These advancements have enabled the incorporation of phylogenetics and genomic epidemiology into the understanding of clinical outbreaks. We show that genomic epidemiology can help to explore the genetic evolution of a pathogen in real time, enabling the identification of the index case and helping understand its transmission dynamics to develop better strategies to prevent future spread of SARS-CoV-2 in congregate, clinical settings such as hospitals.

Corrigendum: Chlamydia pneumoniae CdsL regulates CdsN ATPase activity, and disruption with a peptide mimetic prevents bacterial invasion.

[This corrects the article DOI: 10.3389/fmicb.2011.00021.].

Investigating the impact of quarantine on mental health: insights from the COVID-19 international border surveillance study in Canada.

BACKGROUND: Nations throughout the world are imposing mandatory quarantine on those entering the country. Although such measures may be effective in reducing the importation of COVID-19, the mental health implications remain unclear. AIMS: This study sought to assess mental well-being and factors associated with changes in mental health in individuals subject to mandatory quarantine following travel. METHOD: Travellers arriving at a large, urban international airport completed online questionnaires on arrival and days 7 and 14 of mandated quarantine. Questionnaire items, such as travel history, mental health, attitudes toward COVID-19, and protection behaviours, were drawn from the World Health Organization Survey Tool for COVID-19. RESULTS: There was a clinically significant decline in mental health over the course of quarantine among the 10 965 eligible participants. Poor mental health was reported by 5.1% of participants on arrival and 26% on day 7 of quarantine. Factors associated with a greater decline in mental health were younger age, female gender, negative views toward quarantine measures and engaging in fewer COVID-19 prevention behaviours. For instance, travellers who stated that they rarely wore masks had nearly three times higher odds of developing poor mental health. CONCLUSIONS: Although the widespread use of quarantine may be effective in limiting the spread of COVID-19, the mental health implications are profound and have largely been ignored in policy decisions. Psychiatry has a role to play in contributing to the public policy debate to ensure that all aspects of health and well-being are reflected in decisions to isolate people from others.

COVID-19 international border surveillance at Toronto's Pearson Airport: a cohort study.

OBJECTIVES: The primary objective was to estimate the positivity rate of air travellers coming to Toronto, Canada in September and October 2020, on arrival and on day 7 and day 14. The secondary objectives were to estimate the degree of risk based on country of origin and to assess knowledge and attitudes towards COVID-19 control measures and subjective well-being during the quarantine period. DESIGN: Prospective cohort of arriving international travellers. SETTING: Toronto Pearson Airport Terminal 1, Toronto, Canada. PARTICIPANTS: Participants of this study were passengers arriving on international flights. Inclusion criteria were those aged 18 or older who had a final destination within 100 km of the airport, spoke English or French, and provided consent. Excluded were those taking a connecting flight, had no internet access, exhibited symptoms of COVID-19 on arrival or were exempted from quarantine. MAIN OUTCOME MEASURES: Positive for SARS-CoV-2 virus on reverse transcription PCR with self-administered oral-nasal swab and general well-being using the WHO-5 Well-being Index. RESULTS: Of 16 361 passengers enrolled, 248 (1.5%, 95% CI 1.3% to 1.7%) tested positive. Of these, 167 (67%) were identified on arrival, 67 (27%) on day 7, and 14 (6%) on day 14. The positivity rate increased from 1% in September to 2% in October. Average well-being score declined from 19.8 (out of a maximum of 25) to 15.5 between arrival and day 7 (p<0.001). CONCLUSIONS: A single arrival test will pick up two-thirds of individuals who will become positive by day 14, with most of the rest detected on the second test on day 7. These results support strategies identified through mathematical models that a reduced quarantine combined with testing can be as effective as a 14-day quarantine.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

Detection of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in outpatients: A multicenter comparison of self-collected saline gargle, oral swab, and combined oral-anterior nasal swab to a provider collected nasopharyngeal swab.

BACKGROUND: Widespread testing for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) is necessary to curb the spread of coronavirus disease 2019 (COVID-19), but testing is undermined when the only option is a nasopharyngeal swab. Self-collected swab techniques can overcome many of the disadvantages of a nasopharyngeal swab, but they require evaluation. METHODS: Three self-collected non-nasopharyngeal swab techniques (saline gargle, oral swab and combined oral-anterior nasal swab) were compared to a nasopharyngeal swab for SARS-CoV-2 detection at multiple COVID-19 assessment centers in Toronto, Canada. The performance characteristics of each test were assessed. RESULTS: The adjusted sensitivity of the saline gargle was 0.90 (95% CI 0.86-0.94), the oral swab was 0.82 (95% CI, 0.72-0.89) and the combined oral-anterior nasal swab was 0.87 (95% CI, 0.77-0.93) compared to a nasopharyngeal swab, which demonstrated a sensitivity of ˜90% when all positive tests were the reference standard. The median cycle threshold values for the SARS-CoV-2 E-gene for concordant and discordant saline gargle specimens were 17 and 31 (P < .001), for the oral swabs these values were 17 and 28 (P < .001), and for oral-anterior nasal swabs these values were 18 and 31 (P = .007). CONCLUSIONS: Self-collected saline gargle and an oral-anterior nasal swab have a similar sensitivity to a nasopharyngeal swab for the detection of SARS-CoV-2. These alternative collection techniques are cheap and can eliminate barriers to testing, particularly in underserved populations.

Pandemic Planning in Homeless Shelters: A Pilot Study of a Coronavirus Disease 2019 (COVID-19) Testing and Support Program to Mitigate the Risk of COVID-19 Outbreaks in Congregate Settings.

We tested 104 residents and 141 staff for coronavirus disease 2019 who failed daily symptom screening in homeless shelters in Hamilton, Canada. We detected 1 resident (1%), 7 staff (5%), and 1 case of secondary spread. Shelter restructuring to allow physical distancing, testing, and isolation can decrease outbreaks in shelters.

Comparison of four COVID-19 screening strategies to facilitate early case identification within the homeless shelter population: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: 1. To compare the effectiveness of four different surveillance strategies in detecting COVID-19 within the homeless shelter population. 2. To assess the participant adherence over time for each surveillance method. TRIAL DESIGN: This is a prospective cluster-randomized study to compare the effectiveness of four different surveillance regimens across eight homeless shelters in the city of Hamilton. PARTICIPANTS: Participants will include both residents of, and the staff working within, the homeless shelters. All participants aged 18 or older who consent to the study and are able to collect a swab sample (where relevant) are eligible for the study. The study will take place across eight homeless shelters (four men-only and four women-only) in the City of Hamilton in Ontario, Canada. INTERVENTION AND COMPARATOR GROUPS: The comparator group will receive active daily surveillance of symptoms and testing will only be completed in symptomatic participants (i.e. those who fail screening or who seek care for potential COVID-19 related symptoms). The three intervention arms will all receive active daily surveillance of symptoms and testing of symptomatic participants (as in the comparator group) in addition to one of the following: 1. Once weekly self-collected oral swabs (OS) regardless of symptoms using written and visual instructions. 2. Once weekly self-collected oral-nares swab (O-NS) regardless of symptoms using written and visual instructions. 3. Once weekly nurse collected nasopharyngeal swab (NPS) regardless of symptoms. Participants will follow verbal and written instructions for the collection of OS and O-NS specimens. For OS collection, participants are instructed to first moisten the swab on their tongue, insert the swab between the cheek and the lower gums and rotate the swab three times. This is repeated on the other side. For O-NS collection, after oral collection, the swab is inserted comfortably (about 2-3 cm) into one nostril, parallel to the floor and turned three times, then repeated in the other nostril. NPS specimens were collected by the nurse following standard of care procedure. All swabs were placed into a viral inactivation medium and transported to the laboratory for COVID-19 testing. Briefly, total nucleic acid was extracted from specimens and then amplified by RT-PCR for the UTR and Envelope genes of SARS-CoV-2 and the human RNase P gene, which is used as a sample adequacy marker. MAIN OUTCOMES: 1. PRIMARY OUTCOME: COVID-19 detection rate, i.e. the number of new positive cases over the study period of 8 weeks in each arm of the study. 2. SECONDARY OUTCOMES: Qualitative assessment of study enrollment over 8 weeks. Percentage of participants who performed 50% or more of the weekly swabs in the intervention arms in the 8 week study period. RANDOMIZATION: We will use a computer-generated random assignment list to randomize the shelters to one of four interventions. Shelters were stratified by gender, and the simple randomization scheme was applied within each stratum. The randomization scheme was created using WinPEPI. BLINDING: This is an open-label study in which neither participants nor assessors are blinded. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): Since we are including our total sample frame, a sample size estimation at the cluster level is not required. However, if we succeed to enroll 50 participants per shelter from 8 shelters (n=400), and the detection rate is 3 times higher in the intervention groups (0.15) than in the comparator groups (0.05), we will have 90% power to detect a statistically significant and clinically important difference at a type I error rate of alpha=0.05 (one tailed), assuming an intraclass correlation of ~0.008. These computations were done using WinPEPI, and informed by conservative estimates from other studies on respiratory illness in the homeless (see Full protocol). TRIAL STATUS: The protocol version number is 3.0. Recruitment began on April 17, 2020 and is ongoing. Due to low numbers of COVID cases in the community and shelter system during the initial study period, the trial was extended. The estimated date for the end of the extended recruitment period is Feb 1, 2021. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov on June 18, 2020 with the identifier NCT04438070 . FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Update on Immunotherapeutics in the Management of Metastatic Melanoma

Today, a number of treatment options are now available for metastatic melanoma. Within the last decade, the development of novel immunotherapies for cancer has significantly altered the course of the disease in patients with melanoma. With more patients receiving these potentially life-saving treatments, not only have we learned more about the interplay between the immune system and melanoma, but more importantly, which treatment options are most appropriate given the clinical picture.

Sputum autoantibodies in patients with severe eosinophilic asthma.

BACKGROUND: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. OBJECTIVES: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. METHODS: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. RESULTS: We report a "polyclonal" autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. CONCLUSION: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

Considerations for the rational design of a Chlamydia vaccine.

Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Remarkable progress in vaccine research over the past six decades has led to the advancement of novel C. trachomatis vaccine candidates into clinical trials. However, many questions regarding the role of specific cellular populations and molecular mechanisms in protective immunity against human C. trachomatis genital tract infections remain unanswered. Biomarkers of vaccine induced protective immunity are elusive in humans, while a cautionary message on the translatability of data obtained from current animal models has emanated from vaccine research and development efforts against other important human pathogens. In this commentary, we highlight recent advances in Chlamydia vaccine development and discuss their implications in the context of a rational approach to the design of a human C. trachomatis vaccine.

Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge.

Chlamydia trachomatis infections in women are often asymptomatic and if left untreated can lead to significant late sequelae including pelvic inflammatory disease and tubal factor infertility. Vaccine development efforts over the past three decades have been unproductive and there is no vaccine approved for use in humans. The existence of serologically distinct strains or serovars of C. trachomatis mandates a vaccine that will provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of both structural and effector proteins which is an essential virulence factor for infection and intracellular replication. In this study we evaluated a novel fusion protein antigen (BD584) which consists of three T3SS proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydial vaccine candidate. Intranasal immunization with BD584 elicited serum neutralizing antibodies that inhibited C. trachomatis infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding from the vagina at the peak of infection and cleared the infection sooner than control mice. Immunization with BD584 also reduced the rate of hydrosalpinx by 87.5% compared to control mice. Together, these results suggest that highly conserved proteins of the chlamydial T3SS may represent good candidates for a Chlamydia vaccine.

Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

BACKGROUND: Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS. RESULTS: In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells. CONCLUSION: The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.

Chlamydia pneumoniae CopD translocator protein plays a critical role in type III secretion (T3S) and infection.

Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120-125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.

Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity.

BACKGROUND: Rapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR. OBJECTIVE: To develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction. STUDY DESIGN: We designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens. RESULTS: Optimized M-LAMP was rapid with a mean amplification time of 12 min (compared with 90-120 min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A/H1 and A/H3 from influenza B by Tm. M-LAMP detected 26/28 influenza A/H1, 27/27 influenza A/H3 and 39/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92/94) and 100% (108/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result. CONCLUSIONS: M-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30 min.

Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes.

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.