Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

SARS-CoV-2 and COVID-19: A genetic, epidemiological, and evolutionary perspective.

In less than five months, COVID-19 has spread from a small focus in Wuhan, China, to more than 5 million people in almost every country in the world, dominating the concern of most governments and public health systems. The social and political distresses caused by this epidemic will certainly impact our world for a long time to come. Here, we synthesize lessons from a range of scientific perspectives rooted in epidemiology, virology, genetics, ecology and evolutionary biology so as to provide perspective on how this pandemic started, how it is developing, and how best we can stop it.

Transcriptome analysis of pig intestinal cell monolayers infected with Cryptosporidium parvum asexual stages.

BACKGROUND: Human cryptosporidiosis is caused primarily by two species of apicomplexan protozoa, Cryptosporidium parvum and C. hominis. In cultured cell monolayers, the parasite undergoes two generations of asexual multiplication (merogony). However, the proportion of parasites completing the life-cycle is low and insufficient to sustain continuous propagation. Due to the intracellular location of meronts and later life-cycle stages, oocyst and sporozoites are the only forms of the parasite that can readily be isolated. RESULTS: Research on the replicating forms of Cryptosporidium parasites and their interaction with the host cell remains challenging. Based on an RNA-Seq analysis of monolayers of pig epithelial cells infected with C. parvum, here we report on the impact of merogony on the host's gene regulation. Analysis of the transcriptome of infected and uninfected monolayers demonstrates a significant impact of the infection on host cell gene expression. A total of 813 genes were differentially expressed. Functional terms significantly altered in response to infection include phosphoprotein, RNA binding and acetylation. Upregulation of cell cycle pathways indicates an increase in mitosis. Notably absent from differentially enriched functional categories are stress- and apoptosis-related functions. The comparison of the combined host-parasite transcriptome reveals that C. parvum gene expression is less diverse than the host cell transcriptome and is highly enriched for genes encoding ribosomal functions, such as ribosomal proteins. CONCLUSIONS: These results indicate that C. parvum infection significantly changes host biological functions and provide new insight into gene functions driving early C. parvum intracellular development.

Molecular epidemiology of Cryptosporidium species in livestock in Ireland.

Cryptosporidium is a protozoan that can cause gastro-intestinal illness with diarrhoea in a wide range of hosts. In fact some species of Cryptosporidium can infect the broad range of hosts. The current paper is focused to investigate monthly prevalence and diversity of Cryptosporidium spp. during the spring and early summer (March-June) in 2009 and 2010 in farms with no history of cryptosporidiosis. Animal samples were analyzed to elucidate the prevalence of Cryptosporidium in two regions, West and the East catchments in Ireland. Our investigation demonstrates the prevalence ranges from 14% to 26% an early summer peak (June) was observed. Based on the findings of this study Cryptosporidium ryanae (in cattle, horses), and Cryptosporidium bovis/xiaoi followed by Cryptosporidium parvum (in sheep) were found to be the predominant species in asymptomatic cases. The circulation of other Cryptosporidium species such as C. parvum, C. bovis, C. ubiquitum, C. andersoni and Cryptosporidium horse and pig genotypes in livestock was investigated.