Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would ...significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method’s utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.
In Australia, amoebiasis is thought to occur in travellers, immigrants from endemic areas, and among men who have sex with men. Prevalence of amoebiasis in communities with immigrants from
-endemic ...countries is unknown. The present study is a retrospective case series analysis of patients with laboratory-confirmed amoebiasis from Western Sydney Local Health District, Australia, between years 2005 and 2016. Forty-nine patients with amoebiasis were identified, resulting in an estimated annual incidence of up to 1.1 cases per 100,000 adults. Many were born in Australia (15/47) and India (12/47). Three patients (3/37) had no history of overseas travel, two others had not travelled to an endemic country, and an additional two had a very remote history of overseas travel; one died of fulminant amoebic colitis. Three patients (3/16) were employed in the food industry and one had a history of colonic irrigation in an Australian 'wellness clinic'. Patients had invasive amoebiasis with either liver abscess (41/48) or colitis (7/48), diagnosed most commonly by serology. Invasive procedures were common, including aspiration of liver abscess (28/41), colonoscopy (11/49), and partial hepatectomy (1/49). Although rare, local acquisition of amoebiasis occurs in Western Sydney and contributes to significant morbidity and hospital admissions.
Abstract Inherited rippling muscle disease is an autosomal dominant disorder usually associated with caveolin-3 mutations. Rare cases of acquired rippling muscle disease with abnormal caveolin-3 ...localisation have been reported, without primary caveolin-3 mutations and in association with myasthenia gravis and acetylcholine receptor autoantibodies, or thymoma. We present three new patients with electrically-silent muscle rippling and abnormal caveolin-3 localisation, but without acetylcholine receptor autoantibodies, or clinical or electrophysiological evidence of myasthenia gravis. An autoimmune basis for rippling muscle disease is supported by spontaneous recovery and normalisation of caveolin-3 staining in one patient and alleviation of symptoms in response to plasmapheresis and immunosuppression in another. These patients expand the autoimmune rippling muscle disease phenotype, and suggest that autoantibodies to additional unidentified muscle proteins result in autoimmune rippling muscle disease.
The 2022 seasonal respiratory syncytial virus (RSV) epidemic in Sydney, Australia saw an unprecedented number of RSV detections. We aimed to characterize genomic and immunologic factors associated ...with the surge in RSV cases.
Whole genome sequences of RSV were generated from 264 RSV-infected infants and linked to case-matched clinical data from the 2022 southern hemisphere RSV season. We then performed an immunologic analysis of baseline RSV-specific humoral immunity in women of childbearing age before and throughout the coronavirus disease 2019 pandemic.
Clinical analysis revealed a high burden of disease across patients of all health backgrounds. More than one-half of RSV-related health care visits by infants resulted in hospitalization, and one-quarter required high-flow respiratory support or a higher level of care. Viral phylogenetic analyses revealed that 2022 Sydney RSV sequences were closely related to viruses that had been circulating globally since 2017, including those detected in recent US outbreaks. Nonsynonymous mutations within the palivizumab and nirsevimab binding sites were detected at low frequencies. There was no difference in baseline RSV-neutralizing antibody titers between 2020 and 2022.
Collectively, these findings suggest that neither the emergence of a novel RSV genotype nor hypothesized immune debt was associated with the surge of RSV cases and hospitalizations in 2022. Continued genomic and immunologic surveillance is required to further understand the factors driving outbreaks of RSV globally, and to inform guidelines for the rollout and ongoing use of recently developed immunotherapeutics and vaccines.
Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients ...fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary l-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that l-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.
Specific mutations within the α-skeletal actin gene (ACTA1) result in intranuclear rod myopathy (IRM), characterized by rod-like aggregates containing actin and α-actinin-2 inside the nucleus of ...muscle cells. The mechanism leading to formation of intranuclear aggregates containing sarcomeric proteins and their impact on cell function and contribution to disease pathogenesis is unknown. In this study, we transfected muscle and non-muscle cells with mutants of α-skeletal actin (Val163Leu, Val163Met) associated with intranuclear rod myopathy. By live-cell imaging we demonstrate that nuclear aggregates of actin form within the nuclear compartment, rather than entering the nucleus after formation in the cytoplasm, and are highly motile and dynamic structures. Thus, the nuclear environment supports the polymerization of actin and the movement and coalescence of the polymerized actin into larger structures. We show that the organization of actin within these aggregates is influenced by the binding of α-actinin, and that α-actinin is normally present in the nucleus of muscle and non-muscle cells. Furthermore, we demonstrate that, under conditions of cell stress (cytoskeletal disruption and ATP depletion), WT skeletal actin forms aggregates within the nucleus that are similar in morphology to those formed by the mutant actin, suggesting a common pathogenic mechanism for aggregate formation. Finally, we show that the presence of intranuclear actin aggregates significantly decreases the mitotic index and hence impacts on the function of the cell. Intranuclear aggregates thus likely contribute to the pathogenesis of muscle weakness in intranuclear rod myopathy.
In Australia, amoebiasis is thought to occur in travellers, immigrants from endemic areas,and among men who have sex with men. Prevalence of amoebiasis in communities with immigrants from 'Entamoeba ...histolytica'-endemic countries is unknown. The present study is a retrospective case series analysis of patients with laboratory-confirmed amoebiasis from Western Sydney Local Health District, Australia, between years 2005 and 2016. Forty-nine patients with amoebiasis were identified,resulting in an estimated annual incidence of up to 1.1 cases per 100,000 adults. Many were born in Australia (15/47) and India (12/47). Three patients (3/37) had no history of overseas travel, two others had not travelled to an endemic country, and an additional two had a very remote history of overseas travel; one died of fulminant amoebic colitis. Three patients (3/16) were employed in the food industry and one had a history of colonic irrigation in an Australian 'wellness clinic'. Patients had invasive amoebiasis with either liver abscess (41/48) or colitis (7/48), diagnosed most commonly by serology.Invasive procedures were common, including aspiration of liver abscess (28/41), colonoscopy (11/49),and partial hepatectomy (1/49). Although rare, local acquisition of amoebiasis occurs in Western Sydney and contributes to significant morbidity and hospital admissions.
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