The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world. MAINThe ongoing COVID-19 pandemic has put exceptional strain on public health laboratories, hospital laboratories, and commercial laboratories as they attempt to keep up with demands for SARS-CoV-2 testing. The current diagnostic testing methods recommended by the Centers for Disease Control and Prevention (CDC) in the United States and the World Health Organization (WHO) are traditional RT-qPCR assays that require two steps: first, an RNA extraction from patient nasopharyngeal (NP) swab material, followed by RT-qPCR amplification of the extracted RNA to detect viral RNA 1-3 . The major bottleneck to widespread SARS-CoV-2 testing lies at the RNA extraction step. The simplest manual kit (the Qiagen Viral RNA Mini) is no longer available, and reagents and supplies for the larger automated instruments are extremely limited with uncertain supply chains. While substitution of other RNA extraction kits 4,5 is possible, they too are in limited supply. The current bottleneck is not simply the availability of RNA extraction kits, but also the cost of the extraction assay, the labor and time required to perform it, and the fact that it is rate limiting compared to the downstream RT-qPCR analysis. To address this issue, we tested the unconventional approach of skipping the RNA extraction step altogether and directly loading patient swab material into the RT-qPCR mix. Herein, we report that this approach (which we refer to hereafter as "direct RT-qPCR") correctly identified 92% of samples (n =155) previously shown to be positive for SARS-CoV-2 RNA by conventional RT-qPCR featuring an RNA extraction. Thus, our results suggest that this streamlined assay could greatly alleviate constraints to COVID-19 testing in many regions of the world.

Clostridium difficile is a spore-forming obligate anaerobe that is a leading cause of healthcare-associated infections. C. difficile infections begin when its metabolically dormant spores germinate in the gut of susceptible individuals. Binding of bile salt germinants to the Csp family pseudoprotease CspC triggers a proteolytic signaling cascade consisting of the Csp family protease CspB and the cortex hydrolase SleC. Conserved across the Clostridia, Csp proteases are subtilisin-like serine proteases that activate pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the protective cortex layer, allowing spores to resume metabolism and growth. This signaling pathway, however, is differentially regulated in C. difficile, since CspC functions both as a germinant receptor and regulator of CspB activity. CspB is also produced as a fusion to a catalytically inactive CspA domain that subsequently undergoes interdomain processing during spore formation. In this study, we investigated the role of the CspA pseudoprotease domain in regulating C. difficile spore germination. Mutational analyses revealed that the CspA domain controls CspC germinant receptor levels in mature spores and is required for optimal spore germination, particularly when CspA is fused to the CspB protease. During spore formation, the YabG protease separates these domains, although YabG itself is dispensable for germination. Bioinformatic analyses of Csp family members suggest that the CspC-regulated signaling pathway characterized in C. difficile is conserved in related Peptostreptococcaceae family members but not in the Clostridiaceae or Lachnospiraceae. Our results indicate that pseudoproteases play critical roles in regulating C. difficile spore germination and highlight that diverse mechanisms control spore germination in the Clostridia.

The transition period in dairy cows refers to the period from 3 wk before calving to 3 wk post-calving and is a critical time for influencing milk production and cow health. We hypothesize that the ruminal microbiome shifts as dairy cows transition from a non-lactation period into lactation due to changes in dietary regimen. The purpose of this study was to identify differences in the ruminal microbiome of primiparous and multiparous (study group) cows during the transition period. Five primiparous and 5 multiparous cows were randomly selected from a herd, and ruminal contents were sampled, via stomach tube, 4 times (study day) at 3 wk before calving date (S1), 1 to 3 d post-calving (S2), and 4 (S3) and 8 wk (S4) into lactation and were evaluated for bacterial diversity using 16S pyrotags. Both groups received the same pre-fresh diet (14.6% CP, 44.0% NDF, 21.9% starch) and 3 different lactation diets (L1, L2, and L3) varying in forage base but not amount and formulated to have similar nutrient specifications (16.8% to 17.7% CP; 32.5% to 33.6% NDF; 26.2% to 29.1% starch) post-calving. Forty bacterial communities were analyzed on the basis of annotations of 100,000 reads, resulting in 15,861 operational taxonomic units grouped into 17 bacterial phyla. The UniFrac distance metric revealed that both study group and study day had an effect on the community compositions (P < 0.05; permutational multivariate ANOVA test). The most abundant phyla observed were Bacteroidetes and Firmicutes across all the communities. As the cows transitioned into lactation, the ratio of Bacteroidetes to Firmicutes increased from 6:1 to 12:1 (P < 0.05; Mann-Whitney U test), and this ratio was greater in primiparous cows than in multiparous cows (P < 0.05). This report is the first to explore the effect of parity on dynamics in the ruminal microbiome of cows during the transition period.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples ( n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

BackgroundFungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.ResultsHere, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.ConclusionsWe find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0487-y) contains supplementary material, which is available to authorized users.

Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.

With the COVID‐19 pandemic caused by SARS‐CoV‐2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT–PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g., subgenomic RNA [sgRNA]) and replicative intermediates (e.g., negative‐strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS‐CoV‐2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3 to 37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative‐strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative‐strand, and sgE) detected with the WHO/Charité primer‐probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS‐CoV‐2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cutoff of > 31 or specifically testing for sgRNA can serve as an effective rule‐out test for the presence of culturable virus.

Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.Results: Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.