Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

Biosynthesis and Ether-Bridge Formation in Nargenicin Macrolides.

The nargenicin family of antibiotics are macrolides containing a rare ether-bridged cis-decalin motif. Several of these compounds are highly active against multi-drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron-α-ketoglutarate-dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13-deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides.

Comparative Genomics Shows That Mycobacterium ulcerans Migration and Expansion Preceded the Rise of Buruli Ulcer in Southeastern Australia.

Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans, have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir.IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

Hyperexpression of α-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus.

BACKGROUND: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. RESULTS: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. CONCLUSIONS: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

A Mycobacterium ulcerans vaccine pilot trial using an accurate low-dose challenge.

A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model.

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

Mosquitoes provide a transmission route between possums and humans for Buruli ulcer in southeastern Australia.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.

Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in the walKR 5' untranslated region (5' UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

Outbreak investigation using high-throughput genome sequencing within a diagnostic microbiology laboratory.

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla(CTX-M-15) extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.

Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

Mycobacterium ulcerans not detected by PCR on human skin in Buruli ulcer endemic areas of south eastern Australia.

INTRODUCTION: Mycobacterium ulcerans (MU) causes Buruli ulcer (Buruli), a geographically restricted infection that can result in skin loss, contracture and permanent scarring. Lesion-location maps compiled from more than 640 cases in south eastern Australia suggest biting insects are likely involved in transmission, but it is unclear whether MU is brought by insects to humans or if MU is already on the skin and inoculation is an opportunistic event that need not be insect dependent. METHODS: We validated a PCR swab detection assay and defined its dynamic range using laboratory cultured M. ulcerans and fresh pigskin. We invited volunteers in Buruli-endemic and non-endemic areas to sample their skin surfaces with self-collected skin swabs tested by IS2404 quantitative PCR. RESULTS: Pigskin validation experiments established a limit-of-detection of 0.06 CFU/cm2 at a qPCR cycle threshold (Ct) of 35. Fifty-seven volunteers returned their self-collected kits of 4 swabs (bilateral ankles, calves, wrists, forearms), 10 from control areas and 47 from endemic areas. Collection was timed to coincide with the known peak-transmission period of Buruli. All swabs from human volunteers tested negative (Ct ≥35). CONCLUSIONS: M. ulcerans was not detected on the skin of humans from highly Buruli endemic areas.

Statistical modeling based on structured surveys of Australian native possum excreta harboring Mycobacterium ulcerans predicts Buruli ulcer occurrence in humans.

Background: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. Methods: We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Results: Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SaTScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year's human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. Conclusions: This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU. Funding: This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).

Regulation of the 18 kDa heat shock protein in Mycobacterium ulcerans: an alpha-crystallin orthologue that promotes biofilm formation.

Mycobacterium ulcerans is the causative agent of the debilitating skin disease Buruli ulcer, which is most prevalent in Western and Central Africa. M. ulcerans shares >98% DNA sequence identity with Mycobacterium marinum, however, M. marinum produces granulomatous, but not ulcerative, lesions in humans and animals. Here we report the differential expression of a small heat shock protein (Hsp18) between strains of M. ulcerans (Hsp18(+) ) and M. marinum (Hsp18(-) ) and describe the molecular basis for this difference. We show by gene deletion and GFP reporter assays in M. marinum that a divergently transcribed gene called hspR_2, immediately upstream of hsp18, encodes a MerR-like regulatory protein that represses hsp18 transcription while promoting its own expression. Naturally occurring mutations within a 70 bp segment of the 144 bp hspR_2-hsp18 intergenic region among M. ulcerans strains inhibit hspR_2 transcription and explain the Hsp18(+) phenotype. We also propose a biological role for Hsp18, as we show that this protein significantly enhances bacterial attachment or aggregation during biofilm formation. This study has uncovered a new member of the MerR family of transcriptional regulators and suggests that upregulation of hsp18 expression was an important pathoadaptive response in the evolution of M. ulcerans from a M. marinum-like ancestor.

In vitro activity of SPR719 against Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera.

Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera. We show that SPR719 is active against these NTM species with a MIC range of 0.125-4 µg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rifampicin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.

Population genomics provides insights into the evolution and adaptation to humans of the waterborne pathogen Mycobacterium kansasii.

Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii's success and its ongoing adaptation to the human host.

Identification and Mobilization of a Cryptic Antibiotic Biosynthesis Gene Locus from a Human-Pathogenic Nocardia Isolate.

The genus Nocardia contains >50 human pathogenic species that cause a range of illnesses from skin and soft tissue infections to lung and brain infections. However, despite their membership in the most prominent family of secondary metabolite producers (the Actinomycetes), the ability of Nocardia species, especially those that cause human infections, to produce secondary metabolites has not been as well studied. Using genome mining, we have investigated cryptic secondary metabolite biosynthesis gene clusters from Nocardia species and identified a conserved locus within human pathogenic strains of Nocardia brasiliensis and Nocardia vulneris. Direct capture and heterologous expression in a Streptomyces host activated the biosynthetic locus, revealing it to be the source of the brasiliquinones, benz[a]anthraquinone antibiotics whose biosynthetic pathway has remained hidden for over two decades, until now. Our findings highlight these hitherto neglected human pathogenic Nocardia as a source of diverse and important natural products.

High antibody titres induced by protein subunit vaccines using Mycobacterium ulcerans antigens Hsp18 and MUL_3720 with a TLR-2 agonist fail to protect against Buruli ulcer in mice.

BACKGROUND: Mycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. METHODS: Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. RESULTS: The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. CONCLUSIONS: These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Making time off predictable--and required.

People in professional services believe a 24/7 work ethic is essential for getting ahead--and so they work 60-plus hours a week and stay tethered to their Black-Berrys. This perpetuates a vicious cycle: Responsiveness breeds the need for more responsiveness. When people are always "on," responsiveness becomes ingrained in the way they work, expected by clients and partners, and even institutionalized in performance metrics. There is no impetus to question whether the work actually requires 24/7 responsiveness; on the contrary, people work harder and longer, without stopping to explore how they could work better. But four years of research conducted by the authors in several North American offices of the Boston Consulting Group suggests that consultants and other professionals can provide the highest standards of service and still have planned, uninterrupted time off. They can do this even in times of recession. In this article, Perlow and Porter outline the lessons from BCG's implementation of predictable time off--namely, impose a strict mechanism for taking days and nights off, encourage lots of talk about what's working and what isn't, promote experimentation with different ways of working, and insist on top-level support.