Multi-platform whole genome sequencing for tuberculosis clinical and surveillance applications.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis offers valuable insights for tuberculosis (TB) control. High throughput platforms like Illumina and Oxford Nanopore Technology (ONT) are increasingly used globally, although ONT is known for higher error rates and is less established for genomic studies. Here we present a study comparing the sequencing outputs of both Illumina and ONT platforms, analysing DNA from 59 clinical isolates in highly endemic TB regions of Thailand. The resulting sequence data were used to profile the M. tuberculosis pairs for their lineage, drug resistance and presence in transmission chains, and were compared to publicly available WGS data from Thailand (n = 1456). Our results revealed isolates that are predominantly from lineages 1 and 2, with consistent drug resistance profiles, including six multidrug-resistant strains; however, analysis of ONT data showed longer phylogenetic branches, emphasising the technologies higher error rate. An analysis incorporating the larger dataset identified fifteen of our samples within six potential transmission clusters, including a significant clade of 41 multi-drug resistant isolates. ONT's extended sequences also revealed strain-specific structural variants in pe/ppe genes (e.g. ppe50), which are candidate loci for vaccine development. Despite some limitations, our results show that ONT sequencing is a promising approach for TB genomic research, supporting precision medicine and decision-making in areas with less developed infrastructure, which is crucial for tackling the disease's significant regional burden.

Testing methods to estimate population size for wastewater treatment plants using census data: Implications for wastewater-based epidemiology.

In wastewater-based epidemiology (WBE), wastewater loads are commonly reported as a per capita value. Census population counts are often used to obtain a population size to normalise wastewater loads. However, the methods used to calculate the population size of wastewater treatment plants (WWTPs) from census data are rarely reported in the WBE literature. This is problematic because the geographical extents of wastewater catchments and census area units rarely align perfectly with each other and exist at different spatial scales. This complicates efforts to estimate the number of people serviced by WWTPs in these census area units. This study compared four geospatial methods to combine wastewater catchment areas and census area units to calculate the census population size of wastewater treatment plants. These methods were applied nationally to WWTPs across New Zealand. Population estimates varied by up to 73 % between the methods, which could skew comparisons of per capita wastewater loads between sites. Variability in population estimates (relative standard deviation, RSD) was significantly higher in smaller catchments (rs = -0.727, P < .001), highlighting the importance of method selection in smaller sites. Census population estimates were broadly similar to those provided by wastewater operators, but significant variation was observed for some sites (ranging from 42 % lower to 78 % higher, RSD = 262 %). We present a widely applicable method to calculate population size from census, which involves disaggregating census area units by individual properties. The results reinforce the need for transparent reporting to maintain confidence in the comparison of WBE across sites and studies.

New reference genomes to distinguish the sympatric malaria parasites, Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Despite Plasmodium ovale curtisi (Poc) and wallikeri (Pow) being important human-infecting malaria parasites that are widespread across Africa and Asia, little is known about their genome diversity. Morphologically identical, Poc and Pow are indistinguishable and commonly misidentified. Recent rises in the incidence of Poc/Pow infections have renewed efforts to address fundamental knowledge gaps in their biology, and to develop diagnostic tools to understand their epidemiological dynamics and malaria burden. A major roadblock has been the incompleteness of available reference assemblies (PocGH01, PowCR01; ~ 33.5 Mbp). Here, we applied multiple sequencing platforms and advanced bioinformatics tools to generate new reference genomes, Poc221 (South Sudan; 36.0 Mbp) and Pow222 (Nigeria; 34.3 Mbp), with improved nuclear genome contiguity (> 4.2 Mbp), annotation and completeness (> 99% Plasmodium spp., single copy orthologs). Subsequent sequencing of 6 Poc and 15 Pow isolates from Africa revealed a total of 22,517 and 43,855 high-quality core genome SNPs, respectively. Genome-wide levels of nucleotide diversity were determined to be 2.98 × 10-4 (Poc) and 3.43 × 10-4 (Pow), comparable to estimates for other Plasmodium species. Overall, the new reference genomes provide a robust foundation for dissecting the biology of Poc/Pow, their population structure and evolution, and will contribute to uncovering the recombination barrier separating these species.

Impact of immunosuppressive therapy on SARS-CoV-2 mRNA vaccine effectiveness in patients with immune-mediated inflammatory diseases: a Danish nationwide cohort study.

OBJECTIVE: Patients receiving immunosuppressives have been excluded from trials for SARS-CoV-2 vaccine efficacy. Investigation of immunosuppressants' impact on effectiveness of vaccines, particularly in patients with immune-mediated inflammatory diseases (IMID), is therefore required. DESIGN: We performed a nationwide cohort study to assess the risk of COVID-19 infection in vaccinated patients with IMID exposed to immunosuppressives compared with IMID unexposed to immunosuppressives. Exposure to immunosuppressives in the 120 days before receiving the second SARS-CoV-2 mRNA vaccination was assessed. Patients were followed from date of second vaccination and weighted Cox models were used to estimate the risk of infection associated with immunosuppressives. Secondary outcomes included hospitalisation and death associated with a positive SARS-CoV-2 test. Risk of infection by immunosuppressant drug class was also analysed. SETTING: This study used population-representative data from Danish national health registries in the period from 1 January to 30 November 2021. RESULTS: Overall, 152 440 patients were followed over 19 341 person years. Immunosuppressants were associated with a significantly increased risk of infection across IMID (HR: 1.4, 95% CI 1.2 to 1.5), in inflammatory bowel disease (IBD) (HR: 1.6, 95% CI 1.4 to 1.9) and arthropathy (HR: 1.3, 95% CI 1.1 to 1.4) but not psoriasis (HR: 1.1, 95% CI 0.9 to 1.4). Immunosuppressants were also associated with an increased risk of hospitalisation across IMID (HR: 1.4, 95% CI 1.1 to 2.0), particularly in IBD (HR: 2.1, 95% CI 1.0 to 4.1). No significantly increased risk of death in immunosuppressant exposed patients was identified. Analyses by immunosuppressant drug class showed increased COVID-19 infection and hospitalisation with anti-tumour necrosis factor (TNF), systemic corticosteroid, and rituximab and other immunosuppressants in vaccinated patients with IMID. CONCLUSION: Immunosuppressive therapies reduced effectiveness of mRNA SARS-CoV-2 vaccination against infection and hospitalisation in patients with IMID. Anti-TNF, systemic corticosteroids, and rituximab and other immunosuppressants were particularly associated with these risks.

Tumor Necrosis Factor Inhibitors in Inflammatory Bowel Disease and Risk of Immune Mediated Inflammatory Diseases.

BACKGROUND & AIMS: Tumor necrosis factor inhibitors (anti-TNF) are effective therapies for several immune-mediated inflammatory diseases (IMIDs). However, case reports have identified the paradoxical occurrence of IMIDs in patients treated with anti-TNF. We studied the risk of rheumatoid arthritis, psoriasis, and hidradenitis suppurativa after the initiation of anti-TNF therapy for inflammatory bowel disease (IBD). METHODS: We conducted 2 nationwide cohort studies comprising all patients with IBD in Denmark (2005-2018) and France (2008-2018). We obtained individual-level information on exposure to anti-TNF, diagnoses of IMIDs including rheumatoid arthritis, psoriasis, and hidradenitis suppurativa, and potential confounders from healthcare registers in the respective countries. We used Cox models to estimate hazard ratios (HRs) for the association between anti-TNF exposure and IMIDs and then pooled the estimates from the 2 cohorts. To test the robustness of our results, we performed an active comparator analysis of anti-TNF monotherapy vs azathioprine monotherapy. RESULTS: The Danish and French cohorts comprised 18,258 and 88,786 subjects with IBD, respectively, contributing a total of 516,055 person-years of follow-up. Anti-TNF was associated with an increased risk of rheumatoid arthritis, psoriasis, and hidradenitis suppurativa in both the Danish (HR, 1.66; 95% confidence interval [CI], 1.34-2.07) and the French cohort (HR, 1.78; 95% CI, 1.63-1.94), with a pooled HR of 1.76 (95% CI, 1.63-1.91). Anti-TNF was also associated with an increased risk of the outcomes when compared with azathioprine (pooled HR, 2.94; 95% CI, 2.33-3.70). CONCLUSIONS: In 2 nationwide cohorts of IBD patients, anti-TNF therapy was associated with an increased risk of rheumatoid arthritis, psoriasis, and hidradenitis suppurativa.

Olfaction evaluation in dogs with sudden acquired retinal degeneration syndrome.

PURPOSE: To evaluate olfaction in dogs with sudden acquired retinal degeneration syndrome (SARDS) compared with sighted dogs and blind dogs without SARDS as control groups. ANIMALS STUDIED: Forty client-owned dogs. PROCEDURE: Olfactory threshold testing was performed on three groups: SARDS, sighted, and blind/non-SARDS using eugenol as the test odorant. The olfactory threshold was determined when subjects indicated the detection of a specific eugenol concentration with behavioral responses. Olfactory threshold, age, body weight, and environmental room factors were evaluated. RESULTS: Sixteen dogs with SARDS, 12 sighted dogs, and 12 blind/non-SARDS dogs demonstrated mean olfactory threshold pen numbers of 2.8 (SD = 1.4), 13.8 (SD = 1.4), and 13.4 (SD = 1.1), respectively, which correspond to actual mean concentrations of 0.017 g/mL, 1.7 × 10-13 g/mL and 4.26 × 10-13 g/mL, respectively. Dogs with SARDS had significantly poorer olfactory threshold scores compared with the two control groups (p < .001), with no difference between the control groups (p = .5). Age, weight, and room environment did not differ between the three groups. CONCLUSIONS: Dogs with SARDS have severely decreased olfaction capabilities compared with sighted dogs and blind/non-SARDS dogs. This finding supports the suspicion that SARDS is a systemic disease causing blindness, endocrinopathy, and hyposmia. Since the molecular pathways are similar in photoreceptors, olfactory receptors, and steroidogenesis with all using G-protein coupled receptors in the cell membrane, the cause of SARDS may exist at the G-protein associated interactions with intracellular cyclic nucleotides. Further investigations into G-protein coupled receptors pathway and canine olfactory receptor genes in SARDS patients may be valuable in revealing the cause of SARDS.

Medication-Wide Study: Exploring Medication Use 10 Years Before a Diagnosis of Inflammatory Bowel Disease.

INTRODUCTION: There is growing interest in the prediagnostic phase of inflammatory bowel disease (IBD) and in the overlap of IBD with other diseases. We described and compared use of any prescription medication between individuals with and without IBD in a 10-year period preceding diagnosis. METHODS: Based on cross-linked nationwide registers, we identified 29,219 individuals diagnosed with IBD in Denmark between 2005 and 2018 and matched to 292,190 IBD-free individuals. The primary outcome was use of any prescription medication in years 1-10 before IBD diagnosis/matching date. Participants were considered as medication users if they redeemed ≥1 prescription for any medication in the World Health Organization Anatomical Therapeutic Chemical (ATC) main groups or subgroups before diagnosis/matching. RESULTS: The IBD population had a universally increased use of medications compared with the matched population before IBD diagnosis. At 10 years before diagnosis, the proportion of users was 1.1-fold to 1.8-fold higher in the IBD population in 12 of 14 ATC main groups of medication ( P -value < 0.0001). This applied across age, sex, and IBD subtypes, although it was the most pronounced for Crohn's disease (CD). Two years before diagnosis, the IBD population had a steep increase in medication use for several organ systems. When analyzing therapeutic subgroups of medication, the CD population exhibited 2.7, 2.3, 1.9, and 1.9 times more users of immunosuppressants, antianemic preparations, analgesics, and psycholeptics, respectively, than the matched population 10 years before diagnosis ( P -value < 0.0001). DISCUSSION: Our findings demonstrate universally increased medication use years before IBD, especially CD, diagnosis and indicates multiorgan involvement in IBD.

Sulcus intraocular lens implantation in 17 dogs using a modified ab externo technique.

OBJECTIVE: To describe a modified ab externo method of sulcus intraocular lens (IOL) fixation and report outcomes of eyes treated with this approach. PROCEDURES: Records of patients with lens instability or luxation that underwent a lensectomy and sulcus IOL implantation from January 2004 to December 2020 were reviewed. RESULTS: Nineteen eyes of 17 dogs had a sulcus IOL placed via a modified ab externo approach. The median follow-up time was 546 days (range 29-3387 days). Eight eyes (42.1%) developed POH. A total of six eyes (31.6%) developed glaucoma and required medical management long term to control IOP. The IOL position was satisfactory in most cases. Nine eyes developed superficial corneal ulcers within 4 weeks following surgery, all of which healed without complication. At the time of the last follow-up, 17 eyes were visual (89.5%). CONCLUSIONS: The technique described represents a potentially less technically challenging option for sulcus IOL implantation. The success rate and complications are similar to previously described approaches.

Risk of Recurrent Acute Arterial Events Associated With Thiopurines and Anti-Tumor Necrosis Factor in Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) are at increased risk of acute arterial events. Treatment with anti-tumor necrosis factor (anti-TNF) agents has been associated with a protective effect against the first occurrence of acute arterial events, but the impact of treatment with anti-TNF in patients with a previous history of acute arterial events remains unclear. We assessed the effect of anti-TNF and thiopurines on the risk of recurrent acute arterial events in patients with IBD in a nationwide cohort. METHODS: Based on the French nationwide health insurance database, patients with IBD and a previous history of an acute arterial event were followed up from January 1, 2009, until December 31, 2018. The risk of acute arterial event recurrence associated with anti-TNF and thiopurine exposure was assessed using marginal structural Cox proportional hazard models adjusted for baseline and time-varying covariates. RESULTS: A total of 27,185 patients were included. During 121,822 person-years (median follow-up period, 4.0 y), 6865 recurrent acute arterial events occurred (incidence rate per 1000 person-years, 56.4; 95% CI, 55.0-57.7). Exposure to both anti-TNF and thiopurines were associated with a decreased risk of recurrent acute arterial events compared with the absence of exposure to either treatment (hazard ratio, 0.75; 95% CI, 0.63-0.90 and hazard ratio, 0.76; 95% CI, 0.66-0.88, respectively). CONCLUSIONS: In a nationwide cohort study of patients with IBD and a previous history of an acute arterial event, exposure to both anti-TNF and thiopurines were associated with a decreased risk of recurrent acute arterial events.

A next generation targeted amplicon sequencing method to screen for insecticide resistance mutations in Aedes aegypti populations reveals a rdl mutation in mosquitoes from Cabo Verde.

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for disease control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of mosquitoes at low cost, and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including four non-synonymous polymorphisms in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance.

Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification.

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping.

Sero-epidemiological study of arbovirus infection following the 2015-2016 Zika virus outbreak in Cabo Verde.

In November 2015, cases of Zika virus infection were recorded in Cabo Verde (Africa), originating from Brazil. The outbreak subsided after seven months with 7580 suspected cases. We performed a serological survey (n = 431) in Praia, the capital city, 3 months after transmission ceased. Serum samples were screened for arbovirus antibodies using ELISA techniques and revealed seroconverted individuals with Zika (10.9%), dengue (1-4) (12.5%), yellow fever (0.2%) and chikungunya (2.6%) infections. Zika seropositivity was predominantly observed amongst females (70%). Using a logistic model, risk factors for increased odds of Zika seropositivity included age, self-reported Zika infection, and dengue seropositivity. Serological data from Zika and dengue virus assays were strongly correlated (Spearman's rs = 0.80), which reduced when using a double antigen binding ELISA (Spearman's rs = 0.54). Overall, our work improves an understanding of how Zika and other arboviruses have spread throughout the Cabo Verde population. It also demonstrates the utility of serological assay formats for outbreak investigations.

COVID-profiler: a webserver for the analysis of SARS-CoV-2 sequencing data.

BACKGROUND: SARS-CoV-2 virus sequencing has been applied to track the COVID-19 pandemic spread and assist the development of PCR-based diagnostics, serological assays, and vaccines. With sequencing becoming routine globally, bioinformatic tools are needed to assist in the robust processing of resulting genomic data. RESULTS: We developed a web-based bioinformatic pipeline ("COVID-Profiler") that inputs raw or assembled sequencing data, displays raw alignments for quality control, annotates mutations found and performs phylogenetic analysis. The pipeline software can be applied to other (re-) emerging pathogens. CONCLUSIONS: The webserver is available at http://genomics.lshtm.ac.uk/ . The source code is available at https://github.com/jodyphelan/covid-profiler .

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Actinomyces meyeri causing disseminated actinomycosis in the presence of concurrent bronchogenic carcinoma.

We describe the presentation of a 72-year-old woman with concurrent diagnoses of lung adenocarcinoma in conjunction with disseminated Actinomyces meyeri infection; a rare pathogen which can mimic lung cancer both symptomatically and radiologically. The patient was found to have a pelvic mass initially presumed to be cervical metastases-later confirmed to be of xanthogranulomatous inflammatory origin following transvaginal ultrasound-guided biopsy. The pathogenic cause, identified following pleural aspirate, being a fully sensitive A. meyeri infection; treated with prolonged course amoxicillin.

A silicon singlet-triplet qubit driven by spin-valley coupling.

Spin-orbit effects, inherent to electrons confined in quantum dots at a silicon heterointerface, provide a means to control electron spin qubits without the added complexity of on-chip, nanofabricated micromagnets or nearby coplanar striplines. Here, we demonstrate a singlet-triplet qubit operating mode that can drive qubit evolution at frequencies in excess of 200 MHz. This approach offers a means to electrically turn on and off fast control, while providing high logic gate orthogonality and long qubit dephasing times. We utilize this operational mode for dynamical decoupling experiments to probe the charge noise power spectrum in a silicon metal-oxide-semiconductor double quantum dot. In addition, we assess qubit frequency drift over longer timescales to capture low-frequency noise. We present the charge noise power spectral density up to 3 MHz, which exhibits a 1/fα dependence consistent with α ~ 0.7, over 9 orders of magnitude in noise frequency.

Independent assessment of a deep learning system for lymph node metastasis detection on the Augmented Reality Microscope.

Several machine learning algorithms have demonstrated high predictive capability in the identification of cancer within digitized pathology slides. The Augmented Reality Microscope (ARM) has allowed these algorithms to be seamlessly integrated within the pathology workflow by overlaying their inferences onto its microscopic field of view in real time. We present an independent assessment of the LYmph Node Assistant (LYNA) models, state-of-the-art algorithms for the identification of breast cancer metastases in lymph node biopsies, optimized for usage on the ARM. We assessed the models on 40 whole slide images at the commonly used objective magnifications of 10×, 20×, and 40×. We analyzed their performance across clinically relevant subclasses of tissue, including breast cancer, lymphocytes, histiocytes, blood, and fat. Each model obtained overall AUC values of approximately 0.98, accuracy values of approximately 0.94, and sensitivity values above 0.88 at classifying small regions of a field of view as benign or cancerous. Across tissue subclasses, the models performed most accurately on fat and blood, and least accurately on histiocytes, germinal centers, and sinus. The models also struggled with the identification of isolated tumor cells, especially at lower magnifications. After testing, we reviewed the discrepancies between model predictions and ground truth to understand the causes of error. We introduce a distinction between proper and improper ground truth for analysis in cases of uncertain annotations. Taken together, these methods comprise a novel approach for exploratory model analysis over complex anatomic pathology data in which precise ground truth is difficult to establish.