Detection of CRISPR Adaptation

Russian and American scientists have written a recent review on CRISPR technology. To read more about
CRISPR see Dr. Jennifer Doudna’s laboratory here.

AUTHORS:

Anna Shiriaeva 1 2, Ivan Fedorov 1 3, Danylo Vyhovskyi 1, Konstantin Severinov 1 2 4

AUTHOR AFFILIATIONS:

1 Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia.

2 Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, U.S.A.

3 Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.

4 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.

ABSTRACT:

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

INTRODUCTION:

CRISPR-Cas are diverse (two classes, six types [1–3]) prokaryotic adaptive immunity systems that protect cells from phages and other mobile genetic elements (MGEs) [4,5]. They consist of CRISPR arrays and CRISPR-associated cas genes [6,7]. CRISPR arrays are composed of identical or highly similar repeats separated by unique DNA sequences called spacers [6,7]. The total number of spacers in array varies from one to several hundreds [6,8]. The source of the vast majority (∼93%) of spacers remains unknown, they constitute the ‘dark matter' of CRISPR [9]. Most of the remaining spacers map to MGEs and can be regarded as memories of prior encounters that cells store in CRISPR arrays [9]. Upstream of the CRISPR loci, there is an AT-rich sequence called the ‘leader' [7]. CRISPR arrays are transcribed from a promoter located in the leader and the primary transcript is processed into CRISPR RNAs (crRNAs) containing a single spacer and flanking sequences derived from repeats [10–17]. Cas proteins together with crRNAs form effector complexes (Cascade complex in the type I-E system of Escherichia coli) that recognize ‘protospacers' — DNA or, sometimes, RNA sequences, complementary to a crRNA spacer [13,18–20]. Recognition of protospacers in MGEs leads to their destruction [18–20]. CRISPR immunity is built during CRISPR adaptation, a process which entails incorporation of new spacers in the array [4]. New spacers are typically incorporated at the boundary between the leader and the first repeat and, therefore, the chronological order of spacer acquisition matches the inverse order of spacers in the array [4,21,22]. For every acquired spacer, a new copy of repeat is generated [4,21,22]. Two most conserved Cas proteins, Cas1 and Cas2, common to almost all CRISPR-Cas systems, catalyze integration of spacer precursors (prespacers) into arrays [23–25]. Generally, the acquisition of spacers is not specifically targeted to MGEs and thus spacers from cell's own genome can also be acquired [23,26]. This can result in auto-immune response inhibiting cell growth [27–29]. Not surprisingly, CRISPR adaptation is a tightly controlled process that normally proceeds with very low efficiency and can be difficult to detect both in natural settings and in laboratory experiments. Several methods for the detection of CRISPR adaptation have been developed and helped to shed light on molecular mechanisms governing spacer choice. These methods and their limitations are discussed below.

Selection-based methods of detection of CRISPR adaptation in individual cells or clones

An obvious way to detect the acquisition of a new spacer is to amplify the leader-proximal end of CRISPR array with a pair of primers: one matching the leader, and another matching an internal, pre-existing spacer [23,30,31]. Since new spacers are usually incorporated in front of the first, leader-proximal repeat, and result in repeat duplication [4,21–23], detection of PCR-products extended by integral number of spacer-repeat units reveals CRISPR adaptation events. However, since spacer acquisition can be very infrequent, specific selection of adapted cells is required (Figure 1). Examples of such selections include obtaining colonies of BIMs (bacteriophage insensitive mutants) (Figure 1A) [4,21,22,31,32] or PIMs (plasmid interfering mutants) (Figure 1B) [30,31,33]. These methods are cheap and do not require genetic manipulation of cells under study but they are biased towards interference-proficient spacers acquired from MGEs and thus cannot be used to detect spacers that do not lead to interference against MGEs or lead to self-interference due to acquisition of a spacer from cell's own genome (depending on the CRISPR-Cas subtype, when interference is inactivated, such spacers can constitute from 2 to 99% of acquired spacers [23,26,34,35]).

Two powerful experimental systems have been developed to overcome these limitations and increase the sensitivity of the detection of spacer acquisition events [36,37] (Figure 1C,D). Both systems are based on a reporter gene introduced upstream of the leader sequence of a specifically designed miniaturized E. coli CRISPR array. The reporter is transcribed from a promoter located downstream of the array in a direction opposite to the direction of leader-initiated CRISPR array transcription. The resulting mRNA includes a start codon followed by the leader-CRISPR array segment (cloned in reverse orientation) and the sequence of the reporter which does not have a translational start of its own. In cells with unexpanded CRISPR arrays translation of the reporter ORF is prevented due to an in-frame stop codon within the leader. Insertion of an additional 61-bp long unit (33-bp spacer/28-bp repeat) changes the reading frame and allows the synthesis of the reporter leading to either chloramphenicol resistance [36] (Figure 1C) or fluorescence [37] (Figure 1D) of cells that acquired a spacer. Rare chloramphenicol-resistant colonies can be directly screened for CRISPR array expansion by PCR. With the fluorescent protein-based system, live fluorescent microscopy is used to observe and quantify cells that acquired spacers [37]. Though this has not been implemented yet, the use of FACS (fluorescence-activated cell sorting) should allow one to enrich the population of cells with expanded arrays for downstream analysis. With both systems, the acquisition of spacers that carry stop codons located in the reading frame of the reporter remains undetected. Likewise, incorporation of more than one spacer-repeat unit or incorporation of a single non-standard spacer that fails to restore the reporter reading frame will be undetected. Finally, CRISPR-Cas systems where incorporation of a standard spacer-repeat unit does not shift the reading frame (i.e. introduces an insertion whose length is n × 3 bp, where n is an integral number of nucleotides) cannot be studied.

Detection of CRISPR adaptation in cell populations

In early studies of adaptation, the sequences of newly acquired spacers were determined for individual colonies by Sanger sequencing [4,21–23,30–33]. To analyze millions of CRISPR arrays in a single experiment, high-throughput sequencing (HTS) is usually used [38–40]. This allows one to study biases in spacer length, the distribution of corresponding protospacers along different DNA sources and their nucleotide composition [34,35,38–52]. In principle, with sufficient sequencing depth, HTS of total genomic DNA purified from a culture should reveal reads corresponding to expanded arrays [53]. In a model system of E. coli cultures overproducing the Cas1–Cas2 adaptation protein complex and transformed with spacer-sized oligonucleotides, ∼350× genomic coverage allowed to confidently detect CRISPR array expansion that occurred in ∼10% of cells [54]. Moreover, rarer off-target integration events elsewhere in the genome were also detected [54].

While clearly powerful and unbiased, the shotgun sequencing approach requires high sequencing coverage and provides very low (dozens) numbers of reads corresponding to expanded arrays making it unsuitable for studies aimed at qualitative understanding of spacer selection preferences [54]. Therefore, the common strategy is to prepare PCR amplicons of arrays from cultures undergoing CRISPR adaptation and then subject them for HTS [38–40]. Gel-electrophoresis is used to separate amplicons of initial, unexpanded CRISPR arrays (+0) from those that acquired one (+1), two (+2) or more spacer-repeat units (Figure 2A).

The main problem with PCR-based in-culture methods of detection of CRISPR adaptation is their low sensitivity due to more efficient amplification of shorter (and, in most interesting cases, much more abundant) unexpanded CRISPR arrays [55]. In the case of E. coli type I-E system, the standard method allows one to reliably detect expanded arrays amplicons only in cultures which contain, in our experience, at least 5% of adapted cells. Several modifications aimed to increase the sensitivity have been developed. The simplest one relies on amplification with a leader-specific primer and a primer matching a newly acquired spacer whose sequence is known [40]. After calibration to the amount of PCR product amplified from a region outside of CRISPR array and reflecting the total number of DNA molecules in the sample, this method can be used to determine the efficiency of adaptation by qPCR [56]. The obvious drawback of this method is that it requires prior knowledge about acquired spacer(s) and thus cannot be applied to study spacer acquisition in systems with unknown adaptation preferences. However, it is very powerful when studying acquisition from spacer-sized oligonucleotides transformed into cells [51,54,57].

Another modification uses a leader-specific primer and ‘degenerate' primers matching the repeat sequence and containing one extra 3′-end nucleotide (Figure 2B) [42]. The additional position contains, in equal proportions, three nucleotides except for the one complementary to the last nucleotide of the leader. While amplification products are only expected if (i) a spacer has been acquired and (ii) its last nucleotide is different from the last nucleotide of the leader, in practice amplicons from unexpanded arrays are still observed [42]. The method was reported to detect as little as 0.01% of cells with expanded arrays [42]. However, by design, up to ∼25% of acquired spacers remain undetected.

Moreover, the method is effective only when applied to engineered miniaturized CRISPR arrays reduced to just one repeat, since multiple amplification products from unexpanded arrays with multiple repeats are produced which can't be distinguished from amplicons from expanded arrays.

Reamplification of gel-purified amplicons of expanded arrays allows one to increase the sensitivity of detection of CRISPR adaptation in cell cultures [58]. Products of standard amplification are separated by gel-electrophoresis and purified. The reamplification step is repeated until a fragment of expected length becomes clearly visible on the gel (Figure 2C). The use of automated BluePippin system (agarose gel electrophoresis with automated elution for size selection) allows one to improve the quality of separation, reduce contamination from unexpanded arrays amplicons, and increase the reproducibility of analysis. Even when amplicons of expanded arrays are invisible after first electrophoretic separation, DNA extracted from the corresponding position of the gel can be used for reamplification. Depending on the set of primers designed for reamplification (‘internal', ‘degenerate', or ‘repeat-specific'), this method is reported to detect, correspondingly, 1, 0.01, and 0.1% of cells with expanded arrays within E. coli cultures. ‘Internal' primers can be either the same as the ones used during initial amplification, or a leader-specific ‘nested' reamplification primer annealing closer to the array can be used to increase specificity and avoid amplification of non-CRISPR DNA. In either case, amplification of unexpanded arrays co-purified with expanded ones is not suppressed. ‘Degenerate' primers selectively suppress unexpanded array reamplification as described above. Reamplification with ‘repeat-specific' primers relies on the fact that amplicons corresponding to expanded arrays have two repeats after the first PCR step (Figure 2C). Thus, amplification with primers matching the halves of the repeat sequence yields PCR product only for expanded arrays.

The SENECA [59] pipeline selectively amplifies expanded CRISPR arrays. At the heart of the method is the construction of a plasmid-borne CRISPR array with an FaqI endonuclease recognition site immediately downstream of a miniaturized CRISPR ‘array' consisting of a single repeat preceded by the leader sequence (Figure 2D). Unlike most Type II restriction endonucleases, FaqI, a Type II-S enzyme, cleaves DNA outside of its recognition site generating a sticky end. The CRISPR array used in SENECA is designed such that the recognition of the FaqI site leads to cleavage in the upstream repeat. An Illumina adapter with a sticky end complementary to that generated by FaqI is ligated and PCR with a pair of primers, one complementary to repeat and another — to adapter, selectively amplifies expanded arrays, since the initial repeat sequence is lost after the FaqI treatment and thus amplicons from unexpanded arrays are not amplified. While the published SENECA protocol is based on the use of FaqI, other Type II-S restriction endonucleases could conceivably be used in lieu of FaqI.

The complete article is available at the DOI below.

DOI:

 10.1042/BST20190662

Telomere length correlates with subtelomeric DNA methylation in long-term mindfulness practitioners

In a peer-reviewed paper published earlier this year Dr. Mendioroz and Dr. García-Campayo published their results investigating the relationship between DNA methylation changes and the length of telomeres in cells in people following a long-term meditation practice. As I have been committing more seriously to my meditation practice I love knowing the science behind the changes I am making in my body. The authors state:

“Notably, age showed no association with telomere length in the group of long-term meditators.”

Relationships between intergenic (chr4: 1514317–1514621), GPR31, and SERPINB9 DNA methylation levels and telomere length, according to group. The intergenic region (chr4: 1514317–1514621) is in the first row, GPR31 in the second, and SERPINB9 in the third. Long-term meditators are in the first column and controls are in the second. Methylation levels are represented in the horizontal axis (X), and telomeres length in the vertical axis (Y).

AUTHORS:

Maite Mendioroz, 1,2 Marta Puebla-Guedea, 3 Jesús Montero-Marín, 3,4 Amaya Urdánoz-Casado, 1 Idoia Blanco-Luquin, 1 Miren Roldán, 1 Alberto Labarga, 5 and Javier García-Campayo 3,6

AUTHOR AFFILIATIONS:

1 Neuroepigenetics Laboratory, Navarrabiomed Biomedical Research Center- UPNA-Navarra Institute for Health Research (IdiSNA), Pamplona, Navarra, 31008, Spain. maitemendilab@gmail.com.

2 Department of Neurology, Complejo Hospitalario de Navarra, Pamplona, Navarra, 31008, Spain. maitemendilab@gmail.com.

3 Instituto de Investigación Sanitaria de Aragón. Red de Investigación en Atención Primaria (REDIAPP), Zaragoza, Spain.

4 Department of Psychiatry, University of Oxford, Oxford, OX3 7JX, UK.

5 Neuroepigenetics Laboratory, Navarrabiomed Biomedical Research Center- UPNA-Navarra Institute for Health Research (IdiSNA), Pamplona, Navarra, 31008, Spain.

6 Bioinformatics Unit, Navarrabiomed Biomedical Research Center - UPNA-Navarra Institute for Health Research (IdiSNA), Pamplona, Navarra, 31008, Spain.

7 Miguel Servet University Hospital, University of Zaragoza, Zaragoza, Spain.

ABSTRACT:

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions.

DOI: 10.1038/s41598-020-61241-6

Link to full article is here.

It is Time to Address Airborne Transmission of COVID-19

Dr. Lidia Morawska and 240 colleagues submitted a letter for commentary to the World Health Organization (WHO) regarding their expertise to address examples of airborne transmission of SARS-CoV-2. The authors reference various so called superspreader events. One interesting case study reviewed video surveillance at a restaurant where NO direct or indirect contact of individuals with SARS-CoV-2 came into contact with other tables, yet the virus infected other individuals at those tables. The WHO has since included airborne transmission precautions to their list of recommendations for public health guidance. The full commentary and list of contributing authors to the commentary are listed below.

AUTHORS:

Lidia Morawska 1,*, Donald K. Milton 2

COMMENTARY:

We appeal to the medical community and to the relevant national and international bodies to recognize the potential for airborne spread of COVID-19. There is significant potential for inhalation exposure to viruses in microscopic respiratory droplets (microdroplets) at short to medium distances (up to several meters, or room scale), and we are advocating for the use of preventive measures to mitigate this route of airborne transmission. Studies by the signatories and other scientists have demonstrated beyond any reasonable doubt that viruses are released during exhalation, talking, and coughing in microdroplets small enough to remain aloft in air and pose a risk of exposure at distances beyond 1 to 2 m from an infected individual (see e.g. [1-4]). For example, at typical indoor air velocities [5], a 5 μm droplet will travel tens of meters, much greater than the scale of a typical room, while settling from a height of 1.5 m to the floor. Several retrospective studies conducted after the SARS-CoV-1 epidemic demonstrated that airborne transmission was the most likely mechanism explaining the spatial pattern of infections e.g. [6]. Retrospective analysis has shown the same for SARS-CoV-2 [7-10]. In particular, a study in their review of records from a Chinese restaurant, observed no evidence of direct or indirect contact between the three parties [10]. In their review of video records from the restaurant, they observed no evidence of direct or indirect contact between the three parties. Many studies conducted on the spread of other viruses, including respiratory syncytial virus (RSV) [11], Middle East Respiratory Syndrome coronavirus (MERS-CoV) [8], and influenza [2,4], show that viable airborne viruses can be exhaled [2] and/or detected in the indoor environment of infected patients [11-12]. This poses the risk that people sharing such environments can potentially inhale these viruses, resulting in infection and disease. There is every reason to expect that SARS-CoV-2 behaves similarly, and that transmission via airborne microdroplets [10,13] is an important pathway. Viral RNA associated with droplets smaller than 5 μm has been detected in air [14], and the virus has been shown to maintain infectivity in droplets of this size [9]. Other viruses have been shown to survive equally well, if not better, in aerosols compared to droplets on a surface [15]. The current guidance from numerous international and national bodies focuses on hand washing, maintaining social distancing, and droplet precautions. Most public health organizations, including the World Health Organization (WHO) [16], do not recognize airborne transmission except for aerosol-generating procedures performed in healthcare settings. Hand washing and social distancing are appropriate, but in our view, insufficient to provide protection from virus-carrying respiratory microdroplets released into the air by infected people. This problem is especially acute in indoor or enclosed environments, particularly those that are crowded and have inadequate ventilation [17] relative to the number of occupants and extended exposure periods (as graphically depicted in Figure 1). For example, airborne transmission appears to be the only plausible explanation for several superspreading events investigated which occurred under such conditions e.g. [10], and others where recommended precautions related to direct droplet transmissions were followed. The evidence is admittedly incomplete for all the steps in COVID-19 microdroplet transmission, but it is similarly incomplete for the large droplet and fomite modes of transmission. The airborne transmission mechanism operates in parallel with the large droplet and fomite routes, e.g. [16] that are now the basis of guidance. Following the precautionary principle, we must address every potentially important pathway to slow the spread of COVID-19. The measures that should be taken to mitigate airborne transmission risk include:

• Provide sufficient and effective ventilation (supply clean outdoor air, minimize recirculating air) particularly in public buildings, workplace environments, schools, hospitals, and aged care homes.

• Supplement general ventilation with airborne infection controls such as local exhaust, high efficiency air filtration, and germicidal ultraviolet lights.

• Avoid overcrowding, particularly in public transport and public building
  

Such measures are practical and often can be easily implemented; many are not costly. For example, simple steps such as opening both doors and windows can dramatically increase air flow rates in many buildings. For mechanical systems, organizations such as ASHRAE (the American Society of Heating, Ventilating, and Air-Conditioning Engineers) and REHVA (the Federation of European Heating, Ventilation and Air Conditioning Associations) have already provided guidelines based on the existing evidence of airborne transmission. The measures we propose offer more benefits than potential downsides, even if they can only be partially implemented. It is understood that there is not as yet universal acceptance of airborne transmission of SARS-CoV2; but in our collective assessment there is more than enough supporting evidence so that the precautionary principle should apply. In order to control the pandemic, pending the availability of a vaccine, all routes of transmission must be interrupted. We are concerned that the lack of recognition of the risk of airborne transmission of COVID-19 and the lack of clear recommendations on the control measures against the airborne virus will have significant consequences: people may think that they are fully protected by adhering to the current recommendations, but in fact, additional airborne interventions are needed for further reduction of infection risk. This matter is of heightened significance now, when countries are re-opening following lockdowns - bringing people back to workplaces and students back to schools, colleges, and universities. We hope that our statement will raise awareness that airborne transmission of COVID-19 is a real risk and that control measures, as outlined above, must be added to the other precautions taken, to reduce the severity of the pandemic and save lives.

DISCLAIMER:

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any agency/institution.

ACKNOWLEDGEMENT:

Together with the authors, 239 scientists support this Commentary, and their affiliations and contact details are listed in the Supplementary.

The following scientists contributed to formulating this commentary:

Linsey C. Marr, William Bahnfleth, Jose-Luis Jimenez, Yuguo Li, William W. Nazaroff, Catherine Noakes, Chandra Sekhar, Julian Wei-Tze Tang, Raymond Tellier, Philomena M. Bluyssen, Atze Boerstra, Giorgio Buonanno, Junji Cao, Stephanie J. Dancer, Francesco Franchimon, Charles Haworth, Jaap Hogeling, Christina Isaxon, Jarek Kurnitski, Marcel Loomans, Guy B. Marks, Livio Mazzarella, Arsen Krikor Melikov, Shelly Miller, Peter V. Nielsen, Jordan Peccia, Xavier Querol, Olli Seppänen, Shin-ichi Tanabe, Kwok Wai Tham, Pawel Wargocki, Aneta Wierzbicka, Maosheng Yao. The following scientists reviewed the document: Jonathan Abbatt, John Adgate, Alireza Afshari, KangHo Ahn, Francis Allard, Joseph Allen, Celia Alves, Meinrat O. Andreae, Isabella Annesi-Maesano, Ahmet Arısoy, Andrew P. Ault, Gwi-Nam Bae, Gabriel Bekö, Scott C. Bell, Allan Bertram, Mahmood Bhutta, Seweryn Bialasiewicz, Merete Bilde, Tami Bond, Joseph Brain, Marianna Brodach, David M. Broday, Guangyu Cao, Christopher D. Cappa, Annmarie Carlton, Paul K. S. Chan, Christopher Chao, Kuan-Fu Chen, Qi Chen, Qingyan Chen, David Cheong, Per Axcel Clausen, Ross Crawford, Derek Clements-Croome, Geo Clausen, Ian Clifton, Richard L. Corsi, Benjamin J. Cowling, Francesca Romana d'Ambrosio, Ghassan Dbaibo, Richard de Dear, Gianluigi de Gennaro, Peter DeCarlo, Philip Demokritou, Hugo Destaillats, Joanna Domagala-Kulawik, Neil M. Donahue, Caroline Duchaine, Marzenna R. Dudzinska, Dominic E. Dwyer, Greg Evans, Delphine K. Farmer, Kevin P. Fennelly, Richard Flagan, Janine Fröhlich-Nowoisky, Manuel Gameiro da Silva, Christian George, Marianne

The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings

Dr. Rachel Brown and Dr. Michael Zandi, members of the University College of London Queen Square National Hospital for Neurology and Neurosurgery COVID-19 Study Group, have published a peer-reviewed article in the journal Brain. The researchers describe their study of patients with disorders of the brain and nerves, including encephalopathies, encephalitis, and Acute Disseminated Encephalomyelitis, ischemic stroke, and nerve disease and SARS-CoV-2 infection. A link to the full article is available here including the supplementary materials with clinical vignettes from the study population.

As the neuroscientist Dr. Adrian Owen from the Western University in Canada summarizes,

“My worry is that we have millions of people with COVID-19 now. And if in a year’s time we have 10 million recovered people, and those people have cognitive deficits ... then that’s going to affect their ability to work and their ability to go about activities of daily living,”

Here is a link to a message from Dr. Adrian Owen and his lab.

Imaging from Patients 12, 13 and 15 (COVID-19 autoimmune and haemorrhagic encephalitis). Axial MRI from three individuals with para-/post-infectious central syndromes. (A–D) Patient 12: axial fluid-attenuated inversion recovery (FLAIR) images show bilateral hyperintensity in the mesial temporal lobes (A and B), hypothalamus (C) temporal lobes and thalamus (D). (E–H) Patient 13: axial T2-weighted (E), diffusion weighted imaging (DWI) (F), susceptibility weighted imaging (SWI) (G) and post-contrast T1-weighted (H) images show multifocal clusters of lesions involving the deep white matter of both cerebral hemispheres, intralesional cyst-like areas of varied sizes, and some peripheral rims of restricted diffusion (F), some haemorrhagic changes (G), and T1 hypointense ‘black holes’ without contrast enhancement (H). (I–P) Patient 15: axial images at the level of the insula and basal ganglia (I–L) and at the level of the temporal lobes and upper pons (M–P). T2-weighted images (I and M), SWI images (J and N), DWI images (K and O) and contrast-enhanced images (L and P). There are extensive confluent areas of T2 hyperintensity (I and M), with haemorrhagic change on SWI imaging (J and N), restricted diffusion on DWI images (K and O) and peripheral contrast-enhancement (arrows in L and P) in the insular region, basal ganglia and left occipital lobe (I–L) as well as in the medial temporal lobes and upper pons (M–P).

ABSTRACT:

Preliminary clinical data indicate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with neurological and neuropsychiatric illness. Responding to this, a weekly virtual coronavirus disease 19 (COVID-19) neurology multidisciplinary meeting was established at the National Hospital, Queen Square, in early March 2020 in order to discuss and begin to understand neurological presentations in patients with suspected COVID-19-related neurological disorders. Detailed clinical and paraclinical data were collected from cases where the diagnosis of COVID-19 was confirmed through RNA PCR, or where the diagnosis was probable/possible according to World Health Organization criteria. Of 43 patients, 29 were SARS-CoV-2 PCR positive and definite, eight probable and six possible. Five major categories emerged: (i) encephalopathies (n = 10) with delirium/psychosis and no distinct MRI or CSF abnormalities, and with 9/10 making a full or partial recovery with supportive care only; (ii) inflammatory CNS syndromes (n = 12) including encephalitis (n = 2, para- or post-infectious), acute disseminated encephalomyelitis (n = 9), with haemorrhage in five, necrosis in one, and myelitis in two, and isolated myelitis (n = 1). Of these, 10 were treated with corticosteroids, and three of these patients also received intravenous immunoglobulin; one made a full recovery, 10 of 12 made a partial recovery, and one patient died; (iii) ischaemic strokes (n = 8) associated with a prothrombotic state (four with pulmonary thromboembolism), one of whom died; (iv) peripheral neurological disorders (n = 8), seven with Guillain-Barré syndrome, one with brachial plexopathy, six of eight making a partial and ongoing recovery; and (v) five patients with miscellaneous central disorders who did not fit these categories. SARS-CoV-2 infection is associated with a wide spectrum of neurological syndromes affecting the whole neuraxis, including the cerebral vasculature and, in some cases, responding to immunotherapies. The high incidence of acute disseminated encephalomyelitis, particularly with haemorrhagic change, is striking. This complication was not related to the severity of the respiratory COVID-19 disease. Early recognition, investigation and management of COVID-19-related neurological disease is challenging. Further clinical, neuroradiological, biomarker and neuropathological studies are essential to determine the underlying pathobiological mechanisms, which will guide treatment. Longitudinal follow-up studies will be necessary to ascertain the long-term neurological and neuropsychological consequences of this pandemic.

INTRODUCTION:

Since December 2019, almost 10 million cases and 500 000 deaths due to the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been reported worldwide (WHO situation report). Although the respiratory system complications of coronavirus disease 19 (COVID-19) have been the most frequent and life threatening, there are increasing reports of central and peripheral nervous system (PNS) involvement. These neurological complications have included encephalopathy (Helms et al., 2020), meningo-encephalitis (Moriguchi et al., 2020), ischaemic stroke (Beyrouti et al., 2020), acute necrotizing encephalopathy (Poyiadji et al., 2020), and Guillain-Barré Syndrome (GBS) (Toscano et al., 2020). Radiological series have shown infarcts, microhaemorrhages, features of posterior reversible encephalopathy syndrome, or nerve root enhancement (Franceschi et al., 2020; Mahammedi et al., 2020). Zanin and colleagues (2020) have described a case of CNS demyelination post-COVID-19. Detailed neurological assessment and investigation is challenging in those who are critically ill, limiting the opportunity to delineate the underlying pathophysiology and hence, treatment options. The postulated mechanisms of the various neurological syndromes include, either individually or in combination, direct viral neuronal injury (Zubair et al., 2020), a secondary hyperinflammation syndrome (Mehta et al., 2020), para- and post-infectious inflammatory or immune-mediated disorders, or the effects of a severe systemic disorder with the neurological consequences of sepsis, hyperpyrexia, hypoxia, hypercoagulability and critical illness. Here we describe the detailed emerging spectrum of neurological disorders encountered in 43 COVID-19 patients referred to the National Hospital, Queen Square COVID-19 multidisciplinary team meeting (COVID-MDT), run in partnership with infectious disease and virology colleagues at University College London Hospital (UCLH).

MATERIALS AND METHODS:

We reviewed retrospectively the clinical, radiological, laboratory and neuropathological findings from patients referred to the COVID-MDT neurology/encephalitis and neurovascular multi-disciplinary team meetings. The cases summarized were discussed between 9 April and 15 May 2020. Neurological syndromes developing after definite, probable or possible COVID-19, which were likely to be associated with COVID-19 on clinical grounds, were included. Cases for which a more likely alternative pathology was found were excluded. The probability of COVID-19-related neurological disease was determined using WHO criteria [‘Global surveillance for human infection with coronavirus disease (COVID-19)’]: (i) definite (SARS-CoV-2 RNA PCR positive from nasopharyngeal swab, CSF or pathological specimen); (ii) probable (clinical and laboratory features highly suggestive of COVID-19— lymphopenia, raised D-dimer, suggestive chest radiology in the absence of PCR evidence) (Guan et al., 2020); and (iii) possible, in whom temporal or laboratory features indicate an association but another cause was also found (Ellul et al., 2020). Where possible, laboratory results shown are those nearest to onset of neurological symptoms. The classification of the severity of COVID-19 infection was adapted from Wu and McGoogan (2020). Mild disease included patients with non-pneumonia or mild pneumonia, severe disease included patients with dyspnoea and hypoxia requiring supplementary oxygen, and critical disease included patients with respiratory failure requiring assisted ventilation, septic shock, and/or multi-organ dysfunction. Where possible, laboratory results shown are those nearest to onset of neurological symptoms. Consensus clinical criteria were used to classify individuals with specific neurological syndromes including encephalitis (Solomon et al., 2012; Graus et al., 2016), acute demyelinating encephalomyelitis (ADEM) (Pohl et al., 2016), and GBS (Willison et al., 2016). We obtained assent and/or written consent from patients or from their relatives. This study is approved and registered as a service evaluation of our MDT (ref 06-202021-SE) at University College London Hospitals NHS Trust. Some patient details have been submitted for publication as case reports by their treating physicians: Patients 7 and 11 (Khoo et al., in press), Patient 12 (Zambreanu et al., in press), Patient 15 (Dixon et al., 2020), and Patients 36, 37, 39, 41 and 42 (Beyrouti et al., 2020).

DATA AVAILABILITY:

The data that support the findings of this study are available from the corresponding author, upon reasonable request. The data are not publicly available due to ethical restrictions e.g. their containing information that could compromise the privacy of the patients reported.

RESULTS:

The patients included 24 males and 19 females with ages ranging from 16–85 years. Twenty-three of our patients (53%) were non-white. On the basis of a positive nasal-pharyngeal throat SARS-CoV-2 PCR test, 29 were defined as definite COVID-19, eight were probable and six were possible for this association. The severity of the COVID-19 symptoms varied from mild to critical. The patients presented with a wide range of CNS and PNS features including neuroinflammatory diseases and stroke from 6 days before and up to 27 days following the onset of the COVID-19 symptoms. The patients are divided into five categories based on the clinical, neuroradiological, neurophysiological and laboratory features, as summarized in Table 1. We provide a brief summary of the neurological phenotypes in Tables 2–4. Full details of the clinical, viral, immunological, radiological and neurophysiological investigations, management and treatment responses are detailed in the Supplementary material.

The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission

Dr. Valentyn Stadnytskyi from the Laboratory of Chemical Physics in the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health in Bethesda, Maryland and colleagues have studied speech droplets using laser light scattering. The full paper is available here. The authors conclude:

“We estimate that 1 minute of loud speaking generates at least 1,000 virion-containing droplet nuclei that remain airborne for more than 8 minutes. These therefore could be inhaled by others and, according to IAH, trigger a new SARS-CoV-2 infection.”

“The droplet nuclei observed in our present study and previously by APS (2, 9) are sufficiently small to reach the lower respiratory tract, which is associated with an increased adverse disease outcome (19, 20)."

Our laser light scattering method not only provides real-time visual evidence for speech droplet emission, but also assesses their airborne lifetime. This direct visualization demonstrates how normal speech generates airborne droplets that can remain suspended for tens of minutes or longer and are eminently capable of transmitting disease in confined spaces.”

Light scattering observation of airborne speech droplet nuclei, generated by a 25-s burst of repeatedly speaking the phrase “stay healthy” in a loud voice (maximum 85 dBB at a distance of 30 cm; average 59 dBB). (A) Chart of particle count per frame versus time (smoothed with a 24-s moving average), with the red curve representing the top 25% in scattering brightness and the green curve representing the rest. The bright fraction (red) decays with a time constant of 8 min, and the dimmer fraction (green) decays with a time constant of 14 min. Both exponential decay curves return to their respective background level of ca. 0 (red horizontal dashed line) and 0.4 (green dashed line) counts per frame. Time “0” corresponds to the time the stirring fan was turned off. The 25-s burst of speaking started 36 s before time 0. The black arrow (at 0.5 min) marks the start of the exponential fits. (B) Image of the sum of 144 consecutive frames (spanning 6 s) extracted shortly after the end of the 25-s burst of speaking. The dashed circle marks the needle tip used for focusing the camera. The full movie recording is available in ref. 17, with time “0” in the graph at time point 3:38 in the movie.

AUTHORS AND AFFILIATIONS:
Valentyn Stadnytskyi, a Christina E. Bax, b Adriaan Bax, a,1 and Philip Anfinrud a,1

a Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520

b Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104

1 To whom correspondence may be addressed. Email: vog.hin@xab or vog.hin@durnifna.pilihp.

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved May 4, 2020 (received for review April 10, 2020)

Author contributions: C.E.B., A.B., and P.A. designed research; V.S., A.B., and P.A. performed research; V.S. analyzed data; and C.E.B., A.B., and P.A. wrote the paper.

ABSTRACT:

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant air environment, they disappear from the window of view with time constants in the range of 8 to 14 min, which corresponds to droplet nuclei of ca. 4 μm diameter, or 12- to 21-μm droplets prior to dehydration. These observations confirm that there is a substantial probability that normal speaking causes airborne virus transmission in confined environments.

DISCUSSION:

It has long been recognized that respiratory viruses can be transmitted via droplets that are generated by coughing or sneezing. It is less widely known that normal speaking also produces thousands of oral fluid droplets with a broad size distribution (ca. 1 μm to 500 μm) (1, 2). Droplets can harbor a variety of respiratory pathogens, including measles (3) and influenza virus (4) as well as Mycobacterium tuberculosis (5). High viral loads of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been detected in oral fluids of coronavirus disease 2019 (COVID-19)−positive patients (6), including asymptomatic ones (7). However, the possible role of small speech droplet nuclei with diameters of less than 30 μm, which potentially could remain airborne for extended periods of time (1, 2, 8, 9), has not been widely appreciated.

In a recent report (10), we used an intense sheet of laser light to visualize bursts of speech droplets produced during repeated spoken phrases. This method revealed average droplet emission rates of ca. 1,000 s−1 with peak emission rates as high as 10,000 s−1, with a total integrated volume far higher than in previous reports (1, 2, 8, 9). The high sensitivity of the light scattering method in observing medium-sized (10 μm to 100 μm) droplets, a fraction of which remain airborne for at least 30 s, likely accounts for the large increase in the number of observed droplets. Here, we derive quantitative estimates for both the number and size of the droplets that remain airborne. Larger droplets, which are also abundant but associated with close-proximity direct virus transfer or fomite transmission (11), or which can become resuspended in air at a later point in time (12), are not considered here.

According to Stokes’ law, the terminal velocity of a falling droplet scales as the square of its diameter. Once airborne, speech-generated droplets rapidly dehydrate due to evaporation, thereby decreasing in size (13) and slowing their fall. The probability that a droplet contains one or more virions scales with its initial hydrated volume, that is, as the cube of its diameter, d. Therefore, the probability that speech droplets pass on an infection when emitted by a virus carrier must take into account how long droplet nuclei remain airborne (proportional to d−2) and the probability that droplets encapsulate at least one virion (proportional to d3), the product of which is proportional to d.

The amount by which a droplet shrinks upon dehydration depends on the fraction of nonvolatile matter in the oral fluid, which includes electrolytes, sugars, enzymes, DNA, and remnants of dehydrated epithelial and white blood cells. Whereas pure saliva contains 99.5% water when exiting the salivary glands, the weight fraction of nonvolatile matter in oral fluid falls in the 1 to 5% range. Presumably, this wide range results from differential degrees of dehydration of the oral cavity during normal breathing and speaking and from decreased salivary gland activity with age. Given a nonvolatile weight fraction in the 1 to 5% range and an assumed density of 1.3 g⋅mL−1 for that fraction, dehydration causes the diameter of an emitted droplet to shrink to about 20 to 34% of its original size, thereby slowing down the speed at which it falls (1, 13). For example, if a droplet with an initial diameter of 50 μm shrinks to 10 μm, the speed at which it falls decreases from 6.8 cm⋅s−1 to about 0.35 cm⋅s−1. The distance over which droplets travel laterally from the speaker’s mouth during their downward trajectory is dominated by the total volume and flow velocity of exhaled air (8). The flow velocity varies with phonation (14), while the total volume and droplet count increase with loudness (9). Therefore, in an environment of stagnant air, droplet nuclei generated by speaking will persist as a slowly descending cloud emanating from the speaker’s mouth, with the rate of descent determined by the diameter of the dehydrated speech droplet nuclei.

The independent action hypothesis (IAH) states that each virion has an equal, nonzero probability of causing an infection. Validity of IAH was demonstrated for infection of insect larvae by baculovirus (15), and of plants by Tobacco etch virus variants that carried green fluorescent protein markers (16). IAH applies to systems where the host is highly susceptible, but the extent to which IAH is valid for humans and SARS-CoV-2 has not yet been firmly established. For COVID-19, with an oral fluid average virus RNA load of 7 × 106 copies per milliliter (maximum of 2.35 × 109 copies per milliliter) (7), the probability that a 50-μm-diameter droplet, prior to dehydration, contains at least one virion is ∼37%. For a 10-μm droplet, this probability drops to 0.37%, and the probability that it contains more than one virion, if generated from a homogeneous distribution of oral fluid, is negligible. Therefore, airborne droplets pose a significant risk only if IAH applies to human virus transmission. Considering that frequent person-to-person transmission has been reported in community and health care settings, it appears likely that IAH applies to COVID-19 and other highly contagious airborne respiratory diseases, such as influenza and measles.

The output from a green (532 nm) Coherent Verdi laser operating at 4-W optical power was transformed with spherical and cylindrical optics into a light sheet that is ∼1 mm thick and 150 mm tall. This light sheet passed through slits centered on opposite sides of a cubic 226-L enclosure. When activated, a 40-mm, 12-V muffin fan inside the enclosure spatially homogenizes the distribution of particles in the enclosure. A movie showing the arrangement is available (17). Movie clips of speech droplet nuclei were recorded at a frame rate of 24 Hz with high-definition resolution (1,920 × 1,080 pixels). The camera lens provided a horizontal field of view of ∼20 cm. Therefore, the volume intercepted by the light sheet and viewed by the camera is ∼30 cm3. The total number of particles in the enclosure can be approximated by multiplying the average number of particles detected in a single movie frame by the volume ratio of the enclosure to the visualized sheet, which is ∼7,300. Slow convection currents, at speeds of a few centimeters per second, remained for the duration of the recording. These convection currents are attributed to a 0.5 °C temperature gradient in the enclosure (bottom to top) that presumably is due to heat dissipated by the iPhone11 camera, which was attached to the front side of the enclosure. Since the net air flux across any horizontal plane of the enclosure is zero, this convection does not impact the average rate at which droplet nuclei fall to the bottom of the enclosure.

RESULTS AND DISCUSSION:

With the internal circulation fan turned on, the enclosure was purged with HEPA-filtered air for several minutes. Then, the purge shutter was closed, the movie clip was started, the speaker port was opened, and the enclosure was “filled” with speech droplets by someone repeating the phrase “stay healthy” for 25 s. This phrase was chosen because the “th” phonation in the word “healthy” was found to be an efficient generator of oral fluid speech droplets. The internal fan was turned off 10 s after speech was terminated, and the camera continued recording for 80 min. The movie clip was analyzed frame by frame to determine the number of spots/streaks whose maximum single-pixel intensity exceeded a threshold value of 30. Figure. 1 charts the time-dependent decrease in the number of scattering particles detected. We are not yet able to quantitatively link the observed scattered light intensity to the size of the scattering particle because the light intensity varies across the sheet. However, the brightest 25% were found to decay more quickly than the dimmer fraction, with the two curves reasonably well described by exponential decay times of 8 and 14 min, respectively (Figure. 1A). These fits indicate that, near time 0, there were, on average, approximately nine droplet nuclei in the 30-cm3 observation window, with the larger and brighter nuclei (on average) falling to the bottom of the enclosure at faster speeds than the smaller and dimmer ones.

With the assumption that the contents of the box are homogenized by the muffin fan at time 0, the average number of droplets found in a single frame near time 0 corresponds to ca. 66,000 small droplets emitted into the 226-L enclosure, or ca. 2,600 small droplet nuclei per second of speaking. If the particle size distribution were a delta function and the particles were uniformly distributed in the enclosure, the particle count would be expected to remain constant until particles from the top of the enclosure descend to the top of the light sheet, after which the particle count would decay linearly to background level. The observation that the decay profiles are approximately exponential points to a substantial heterogeneity in particle sizes, even after binning them into two separate groups.

The weighted average decay rate (0.085 min−1) of the bright and dim fractions of particles (Fig. 1A) translates into a half-life in the enclosure of ca. 8 min. Assuming this half-life corresponds to the time required for a particle to fall 30 cm (half the height of the box), its terminal velocity is only 0.06 cm⋅s−1, which corresponds to a droplet nucleus diameter of ∼4 μm. At the relative humidity (27%) and temperature (23 °C) of our experiment, we expect the droplets to dehydrate within a few seconds. A dehydrated particle of 4 μm corresponds to a hydrated droplet of ca. 12- to 21-μm diameter, or a total hydrated volume of ∼60 nL to 320 nL for 25 s of loud speaking. At an average viral load of 7 × 106 per milliliter (7), we estimate that 1 min of loud speaking generates at least 1,000 virion-containing droplet nuclei that remain airborne for more than 8 min. These therefore could be inhaled by others and, according to IAH, trigger a new SARS-CoV-2 infection.

The longest decay constant observed by us corresponds to droplets with a hydrated diameter of ≥12 μm when exiting the mouth. The existence of even smaller droplets has been established by aerodynamic particle sizer (APS) measurements (2). APS is widely used for detecting aerosol particulates and is best suited for particles in the 0.5- to 5-μm range. Morawska et al. (2) detected as many as 330 particles per second in the 0.8- to 5.5-μm range upon sustained “aah” vocalization. Considering the short travel time (0.7 s) between exiting the mouth and the APS detector, and the high relative humidity (59%) used in that study, droplet dehydration may have been incomplete. If it were 75% dehydrated at the detector, an observed 5.5-μm particle would have started as an 8.7-μm droplet when exiting the mouth, well outside the 12- to 21-μm range observed above by light scattering. This result suggests that APS and light scattering measurements form a perfect complement. However, we also note that, even while the smallest droplet nuclei effectively remain airborne indefinitely and have half-lives that are dominated by the ventilation rate, at a saliva viral load of 7 × 106 copies per milliliter, the probability that a 1-μm droplet nucleus (scaled back to its originally hydrated 3-μm size) contains a virion is only 0.01%.

Our current setup does not detect every small particle in each frame of the movie, and our reported values are therefore conservative lower limit estimates. We also note that the saliva viral load shows large patient-to-patient variation. Some patients have viral titers that exceed the average titer of Wölfel et al by more than two orders of magnitude (7, 18), thereby increasing the number of virions in the emitted droplets to well over 100,000 per minute of speaking. The droplet nuclei observed in our present study and previously by APS (2, 9) are sufficiently small to reach the lower respiratory tract, which is associated with an increased adverse disease outcome (19, 20).

Our laser light scattering method not only provides real-time visual evidence for speech droplet emission, but also assesses their airborne lifetime. This direct visualization demonstrates how normal speech generates airborne droplets that can remain suspended for tens of minutes or longer and are eminently capable of transmitting disease in confined spaces.

DATA AVAILABILITY STATEMENT:

All raw data used for analysis are available in ref. 17.

Chloroquine and Hydroxychloroquine for COVID-19: A Word of Caution

Dr. Srinadh Annangi, MD has summarized his views and published in the July issue of the Respiratory journal. The full article is included below. He concludes:

“The Hippocratic oath of ‘first do no harm’ should drive our instincts in these uncertain times. Authorities should make recommendations based on clinical evidence and not speculations. There is no conclusive evidence to support these drugs either for treatment or prophylaxis. Even if the results from ongoing trials are more conclusive, ideal dosing regimens should be evaluated to avoid unwarranted overdosing. Until then, CQ and HCQ should only be used in the setting of clinical trials with a clear understanding among physicians and patients of uncertain efficacy and potentially harmful side effects.”

The novel coronavirus, severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), is the causative pathogen of the coronavirus disease 2019 (COVID‐19) pandemic. There is interest in recommending chloroquine (CQ) or hydroxychloroquine (HCQ) for treatment and prophylaxis, as they are readily available and affordable. Shah et al. have recently reviewed the current literature on the use of CQ and HCQ as prophylaxis against COVID‐19. 1 Prior research including with human immunodeficiency virus (HIV) and SARS‐CoV‐1 has shown that in vitro antiviral activity of CQ and HCQ does not necessarily translate to in vivo clinical efficacy. However, the 79% genomic similarity between SARS‐CoV‐1 and ‐2, along with their similar receptor‐binding domain structures, has stimulated interest in CQ and HCQ. 2

Yao et al., in their antiviral pre‐treatment activity analysis, reported EC50 (50% maximal effective concentration) for CQ and HCQ at 24 h as >100 and 6.25 μM, respectively, much higher compared to values from their treatment analysis arm. 3 It is unclear how the differences in the EC50 values will impact the in vivo prophylactic antiviral efficacy at their recommended doses of HCQ 400 mg twice daily (bd) on day 1 and then 200 mg bd for 5 days. Ongoing prophylaxis clinical trials in the United States are using doses ranging from 200 to 400 mg daily or weekly, with or without a loading dose for a total duration of 5 days to 3 months. The results of these studies will only answer the effectiveness, if any, of these dosing regimens.

Analysis of cytotoxic effects is based on CCK8 assays, an indirect colorimetric assay evaluating the number of viable cells by measuring their metabolic activity. However, some of the side effects of CQ and HCQ are based on their inherent pharmacological properties rather than their direct cytotoxic effect. In a recent unpublished study by Chorin et al. from New York evaluating 80 hospitalized SARS‐CoV‐2‐infected patients receiving HCQ + Azithromycin, QTc (corrected QT interval) prolongation by >40 ms was noted in 30% of subjects and 11% had a prolongation to >500 ms. Baseline QTc did not predict the risk for QTc prolongation. 4 Cardiac injury, renal impairment, concomitant use of drugs that prolong QTc and electrolyte imbalance will further increase the risk of fatal ventricular arrhythmias.

Anecdotal reports of death from self‐administered CQ phosphate were reported. Although retinal toxicity is rare at currently recommended doses, risk increases among patients with renal or hepatic impairment with decreased drug elimination. In a recent mice study, using a combination of CQ and HCQ (60 mg/kg daily for 4 weeks) with metformin (250 mg/kg daily for 4 weeks) resulted in 30–40% lethality. 5 The safety profile of this combination at the much lesser doses currently being recommended for prophylaxis is yet to be established, and caution should be excised by prescribers until then. Potential overdose can also happen in obese patients if the dose is calculated using total body weight instead of ideal body weight as these drugs are not retained in the fatty tissue. 6

Published studies to date have not reported prophylactic efficacy of these compounds. Even though Gautret et al. supported the treatment efficacy, it was extensively criticized for study design limitations. 7 Some subjects attained virologic cure as early as Day 1; however, these patients might have attained virologic cure irrespective of treatment and attributing this to the antiviral efficacy of HCQ is not appropriate. Chen et al., in their analysis, did not report virologic cure. 8

The Hippocratic oath of ‘first do no harm’ should drive our instincts in these uncertain times. Authorities should make recommendations based on clinical evidence and not speculations. There is no conclusive evidence to support these drugs either for treatment or prophylaxis. Even if the results from ongoing trials are more conclusive, ideal dosing regimens should be evaluated to avoid unwarranted overdosing. Until then, CQ and HCQ should only be used in the setting of clinical trials with a clear understanding among physicians and patients of uncertain efficacy and potentially harmful side effects.

Respirology 2020 Jul;25(7):683-684.

DOI: 10.1111/resp.13845

REFERENCES:

1. Shah S, Das S, Jain A, Misra DP, Negi VS. A systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease‐19 (COVID‐19). Int. J. Rheum. Dis. 2020; 10.1111/1756-185X.13842 [CrossRef] [Google Scholar]

2. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020; 395: 565–74. [PMC free article] [PubMed] [Google Scholar]

3. Yao X, Ye F, Zhang M, Cui C, Huang B, Niu P, Liu X, Zhao L, Dong E, Song C et al In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Clin. Infect. Dis. 2020. 2020. pii: ciaa237. 10.1093/cid/ciaa237 [CrossRef] [Google Scholar]

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5. Rajeshkumar N, Yabuuchi S, Pai SG, Maitra A, Hidalgo M, Dang CV. Fatal toxicity of chloroquine or hydroxychloroquine with metformin in mice. bioRxiv 2020; 10.1101/2020.03.31.018556 [CrossRef] [Google Scholar]

6. Laaksonen A‐L, Koskiahde V, Juva K. Dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus: a clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine. Scand. J. Rheumatol. 1974; 3: 103–8. [PubMed] [Google Scholar]

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Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection

For those of you that attended the American Association of University Women “FUN”draiser in November, you will not be surprised to find the diagram I shared with the green, double-arrow below connecting Swine and Humans in the transmission of influenza genetic material and the ongoing surveillance studies needed to predict the next possible strains of pathogens transmissible to humans. Two days ago, the Proceedings of the National Academy of Sciences published a peer-reviewed and submitted article sharing surveillance data from 2011-2018. A link to the article and summary of the findings are summarized here.

SIGNIFICANCE:

Pigs are intermediate hosts for the generation of pandemic influenza virus. Thus, systematic surveillance of influenza viruses in pigs is a key measure for pre warning the emergence of the next pandemic influenza. Here, we identified a reassortant EA H1N1 virus possessing pdm/09 and TR-derived internal genes, termed as G4 genotype, which has become predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses have all the essential hallmarks of a candidate pandemic virus. Of concern is that swine workers show elevated seroprevalence for G4 virus.

Controlling the prevailing G4 EA H1N1 viruses in pigs and close monitoring in human populations, especially the workers in swine industry, should be urgently implemented.

AUTHORS:
Honglei Sun, Yihong Xiao, Jiyu Liu, Dayan Wang, Fangtao Li, Chenxi Wang, Chong Li, Junda Zhu, Jingwei Song, Haoran Sun, Zhimin Jiang, Litao Liu, Xin Zhang, Kai Wei, Dongjun Hou, Juan Pu, Yipeng Sun, Qi Tong, Yuhai Bi, Kin-Chow Chang, Sidang Liu, George F. Gao, and Jinhua Liu

ABSTRACT:

Pigs are considered as important hosts or “mixing vessels” for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.

AUTHOR AFFILIATIONS:

Honglei Sun a,1, Yihong Xiao b,1, Jiyu Liu a,1, Dayan Wang c,d, Fangtao Li a, Chenxi Wang a, Chong Li a, Junda Zhu a, Jingwei Song a, Haoran Sun a, Zhimin Jiang a, Litao Liu a, Xin Zhang a, Kai Wei b, Dongjun Hou a, Juan Pu a, Yipeng Sun a, Qi Tong a, Yuhai Bi e, Kin-Chow Chang f, Sidang Liu b, George F. Gao c,d,e,2, and Jinhua Liua,2

a Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, 100193 Beijing, China;

b Department of Fundamental Veterinary Medicine, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 271000 Tai’an, China;

c Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206 Beijing, China;

d World Health Organization Collaborating Center for Reference and Research on Influenza, 102206 Beijing, China;

e Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Center for Influenza Research and Early-Warning, Chinese Academy of Sciences, 100101 Beijing, China;

f School of Veterinary Medicine and Science, University of Nottingham, Loughborough LE12 5RD, United Kingdom

Contributed by George F. Gao, April 28, 2020
(sent for review December 9, 2019; reviewed by Ian H. Brown and Xiu-Feng Henry Wan)

DOI:  

https://doi.org/10.1073/pnas.1921186117

Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak

This study highlights the importance of selecting various primer sets when doing PCR-based testing for the SARS-CoV-2 genome. I used the QUIAGEN platform combining multiple primer sets in 2000-2002 to develop Adenoviral Diagnostic tests using the TaqMan technology newly available at the time. This research is summarized in the following publications.

This research was conducted by members of the QUIAGEN Technical and Quality Control teams listed below. This research is helpful to those interesting in molecular diagnostics in general as well as research and commercial labs conducting SARS-CoV-2 assay panels.

AUTHORS: Luis Penarrubia,a Maria Ruiz,a Roberto Porco,a Sonia N. Rao,b Martí Juanola-Falgarona,a Davide Manissero,c Marta López-Fontanals,a,⁎ and Josep Parejaa,⁎

Objectives

In this study, five SARS-CoV-2 PCR assay panels were evaluated against the accumulated genetic variability of the virus to assess the effect on sensitivity of the individual assays.

Design or methods

As of week 21, 2020, the complete set of available SARS-CoV-2 genomes from GISAID and GenBank databases were used in this study. SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated.

Results

A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay. Variations in 8,773 (25.94%) genomes were considered as high risk, whereas additional 2,854 (8.43%) genomes presented low frequent single mutations and were predicted to yield no impact on sensitivity. In case of the QIAstat-Dx SARS-CoV-2 Panel, 99.11% of the genomes matched with a 100% coverage all oligonucleotides, and critical variations were tested in vitro corroborating no loss of sensitivity.

Conclusions

This analysis stresses the importance of targeting more than one region in the viral genome for SARS-CoV-2 detection to mitigate the risk of loss of sensitivity due to the unknown mutation rate during this SARS-CoV-2 outbreak.

INTRODUCTION

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 (Zhu et al., 2020), the number of confirmed cases has been rising dramatically (The Open Science Prize, 2020). According to data from the World Health Organization (WHO), Coronavirus Disease 2019 or COVID-19 (Gorbalenya et al., 2020) has been diagnosed in over 6,057,853 people from more than 200 countries or territories and caused over 371,166 deaths worldwide as of 1 June 2020 (World Health Organization, 2020a).SARS-CoV-2 is the seventh coronavirus known to infect humans and it appears to have its origin from zoonotic transmission similar to SARS-CoV (2002) and MERS-CoV (2012) (Andersen et al., 2020). SARS-CoV-2 is hypothesised to be the product of recombination or mutation from a genetically related SARS-CoV species hosted by bats (Sun et al., 2020). Unlike seasonal human coronaviruses HKU1, NL63, OC43 and 229E which are associated with mild symptoms (Corman et al., 2018), SARS-CoV-2 can cause pneumonia and severe acute respiratory syndrome (Andersen et al., 2020). All coronaviruses are single, positive-stranded RNA viruses with similar genomic ∼30 kb size and structure. These genes encode non-structural proteins (ORF1a and ORF1b) involved in replication at the 5′-end and multiple structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) downstream that make up the virus particle (Chan et al., 2020; Wu et al., 2020).During novel RNA viral outbreaks, detection of the virus relies on real-time reverse transcription polymerase chain reaction (real-time RT-PCR) for detection of the RNA of the virus In the case of SARS-CoV-2, RT-PCR is used for confirmatory diagnosis of symptomatic patients as well as increasingly screening of asymptomatic contacts and subjects at risk. The unprecedented outbreak posed the challenge of evaluating the different diagnostic RT-PCR assays sensitivity and specificity without a previous established gold standard.Since the availability of the first SARS-CoV-2 genomic sequence (Wu et al., 2020), several studies have reported a rapid genetic evolution of SARS-CoV-2 through a phylogenetic tree tracking the geographical spread of the virus (Andersen et al., 2020; The Open Science Prize, 2020; Wu et al., 2020; Yi, 2020). As previously established for RNA viruses, the nucleotide mutation rate of SARS-CoV-2 is estimated to be 8E-04 substitutions per site per year as a consequence of the lack of proofreading activity of polymerases (Lauring and Andino, 2020; The Open Science Prize, 2020). It evinces that new genetic variations of SARS-CoV-2 that will most likely occur during this evolving outbreak could compromise the sensitivity and specificity of RT-PCR detection (Lippi et al., 2020).Specifically, both natural mutation rate of a virus (Lauring and Andino, 2020) and active viral recombination (Yi, 2020) could potentially impair the efficiency of oligonucleotide annealing (Lippi et al., 2020), affecting sensitivity (increasing the limit of detection) or inclusivity (missed detection of some strains due to genomic changes). We hypothesize that in order to minimize the risk of sensitivity loss of RT-PCR for detecting novel viruses with unknown genetic variability like SARS-CoV-2, published RT-PCR design strategies for SARS-CoV-2 detection should target more than one region in the viral genome. This was the general approach followed to obtain the earliest available SARS-CoV-2 RT-PCR assays in panels used worldwide (Centers for Disease Control and Prevention, 2020a, 2020b; Chu et al., 2020; Corman et al., 2020; National Microbiology Data Center, 2020; QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020; University of Hong Kong, 2020; World Health Organization, 2020b), including the syndromic multiplex QIAstat-Dx Respiratory SARS-CoV-2 Panel (Table 1). The selected conserved target regions from available SARS-CoV-2 genomes minimizes the potential cross-reactivity with other coronaviruses (Gorbalenya et al., 2020; Zhu et al., 2020), including human coronaviruses (Wu et al., 2020) and bat-SARS-like coronaviruses (Chan et al., 2020).The goal of this study is to assess how the genetic variability observed in the SARS-CoV-2 genome as of week 21, 2020 has affected sensitivity of publicly available SARS-CoV-2 real-time RT-PCR Panel assays. Additionally, a detailed characterization and evaluation of the impact on performance of the SARS-CoV-2 assays in the QIAstat-Dx Respiratory SARS-CoV-2 Panel was carried out.

METHODS

The complete set of available SARS-CoV-2 genomes larger than 26,000 nucleotides from GISAID (https://www.gisaid.org/) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) databases were used in this study. As of week 21, 2020, a total of 30,090 and 3,729 available genomes (GISAID and GenBank respectively, Table S1) were aligned using ClustalW algorithm implemented in Geneious software v.10.0.3. (http://www.geneious.com), with a gap open and extent cost of 5 and 3 respectively. The output alignment was curated manually.SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx (Table 1) were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated (Table S2). Variations were considered as high risk when they were placed in the three last nucleotides of the 3’-end, affecting the PCR elongation step (Stadhouders et al., 2010; Whiley and Sloots, 2005) or in the 5’-end of the probe affecting the natural 5’- exonuclease activity of the polymerases (Smith et al., 2002). In addition, were also considered as high risk those variations with a significant presence (>1%) among all published genomes that could become more extended worldwide in the current or potential future spread episodes.

RESULTS

Results showed large nucleotide insertions (EPI_ISL_416672, EPI_ISL_418061, EPI_ISL_418066, EPI_ISL_416720, EPI_ISL_423027, EPI_ISL_424274, EPI_ISL_424327, EPI_ISL_426134, EPI_ISL_427267, EPI_ISL_429646, EPI_ISL_430896, EPI_ISL_434554, EPI_ISL_437867, EPI_ISL_445217) or fragment gene inversions (EPI_ISL_427289, EPI_ISL_427291) mainly in the polyprotein Orf1ab gene, typically indicating genetic recombination common in coronavirus genomes (Sun et al., 2020; Yi, 2020). Since none of them were placed in binding regions of any of the PCR assays, recombination analysis was considered out of the scope of this current study.A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay. Variations in 8,773 (25.94%) genomes were considered as high risk: a) a trinucleotide variation (GGG to AAC) present in 8,251 of the 8,257 (24.41%) genomes affecting the binding of the N gene NMDC assay forward primer; b) different single variations in the last three positions of the 3’-end or 5’-end (primers and probes respectively). Genetic variations affecting annealing of more than one oligonucleotide of the same assay were not frequent: a) a total of 126 out of the 128 genomes with a non-critical mismatch in Reverse primer of the WHO RdRp assay (Table 2) present any other variation in the Probe (RdRp_SARSr-P2) or in the Forward primer binding regions, b) 17 out of the 8,251 genomes with multiple three consecutive mismatches at the 5’-end (GGG to AAC) of the NMDC N assay presented additional single variations in the Probe or Reverse primer binding regions; c) five genome sequences (EPI_ISL_444736 for the WHO N assay; EPI_ISL_437536, EPI_ISL_422983 and MT293178 for the CDC N1 assay; and EPI_ISL_446918 for the CDC N2 assay) presented combinations of non-critical mismatches among different oligonucleotides of the same set.Additional 2,854 (8.44%) genomes presented low frequent single mutations considered to be low risk (Table 2) and were predicted to yield no impact on sensitivity. However, the effect in every PCR performance must be evaluated individually based on the corresponding PCR thermal profile conditions. The impact of non-3 terminal mismatches should not be underestimated, especially in a diagnostic environment (Stadhouders et al., 2010; Whiley and Sloots, 2005).A large number of genomes with variations affecting the CDC N3 assay were also found (546 variations, 1.61%). Although they cannot be classified as critical based on the parameters defined in the present study, this data supports why the initial N3 CDC assay was removed from the official panel due to inconclusive results (Centers of Disease Control and Prevention, 2020a, 2020b).This same method of evaluation was carried out with the QIAstat-Dx SARS-CoV-2 Panel oligonucleotide sequences. The SARS-CoV-2 assay was originally developed as a real-time RT-PCR duplex assay covering two highly conserved target genes (RdRp and E) reported with the same fluorophore and showing a verified combined limit of detection (LoD) of 500 copies/mL in clinical samples (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020). A total of 33,518 (99.11 %) of 33,819 matched with a 100% coverage all oligonucleotides included in the SARS-CoV-2 assays of the QIAstat-Dx Respiratory SARS-CoV-2 Panel. In contrast, 301 (0.89%) genomes showed a single variation affecting annealing for any oligonucleotide, and 35 (0.10%) of those 301 genomes contained a variation in any critical position of any primer or probe end. Among these latter ones, the most frequent variation consisted of a mismatch in the last position of the 3’-end of one of the primers (7 genomes, 0.02%). Because a mismatch in this position might impair the elongation PCR step (Stadhouders et al., 2010; Whiley and Sloots, 2005), this genomic mutation was further tested in vitro re-evaluating the LoD. A total of twenty replicates of quantified synthetic dsDNA reproducing the sequence containing this mutation were run at the LoD concentration (500 copies/mL) described in the publicly available QIAstat-Dx Respiratory SARS-CoV-2 Panel instructions for use (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020), and 19 out of the 20 (95%) replicates were positive (Table S3) confirming the defined LoD. Therefore, the QIAstat-Dx SARS-CoV-2 assay remains highly specific and sensitive against SARS-CoV-2 even in light of genomic variations from the first three months of the COVID-19 outbreak (Visseaux et al., 2020).

DISCUSSION

Genetic variability observed among SARS-CoV-2 genomes available until week 15, 2020 after the onset of the outbreak showed single nucleotide mutations affecting the annealing of all SARS-CoV-2 RT-PCR panels evaluated in this study. The majority of the annotated single nucleotide variations are predicted to have no effect on sensitivity at panel level, including those placed in critical positions. This was confirmed for the QIAstat-Dx SARS-CoV-2 Panel to show no effect on the LoD of the panel.Given that genetic variability in the SARS-CoV-2 genome is expected to increase based on the natural viral mutation and recombination rates, our results show that combination of more than one assay target in real-time RT-PCR SARS-CoV-2 panels can mitigate the risk of loss of sensitivity or specificity. In this regards, continuous monitoring of genomic variations is essential to provide a rapid response in case assay re-design is needed.

Acknowledgements

We thank all those who have contributed sequences to the GISAID database (https://www.gisaid.org/) and to NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). We also thank the QIAstat-Dx Barcelona team for their support in their corresponding roles.

Conflict of interests: 

LP, RP, MR, SNR, MJF,DM,MLF and JP are QIAGEN employees.No ethical approval was required to conduct this work. The work described in this paper was funded by the employer of the authors (QIAGEN).

References

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Authors contributions

LP: Planned experiments, conceptualised the laboratory work, conceptualised the overall study, wrote the manuscript draft.MR: Planned and conducted experiments.RP: Planned and conducted experiments.SR: Contributed to overall study planning and review of the manuscript.MJ: Contributed to overall study planning and review of the manuscript.DM: Contributed to overall study conceptualization and review of the manuscript.MLF: Contributed to overall study planning and review of the manuscript.JP: Contributed to overall study conceptualization, co-wrote of the manuscript.Table 1. Description of earliest available SARS-CoV-2 RT-PCR assaysRT-qPCR panel Assays included Target genes Description fReferences WHO panel a3RdRp, E, Nthe E gene assay is used as the firstline screening tool, then followed by confirmatory testing with an RdRp gene assay. The N gene assay can eventually be analyzed as an additional confirmatory assay.Corman et al., 2020; World Health Organization, 2020bCDC (US) panelb2NThis panel contains two monoplex assays (N1, N2) designed for specific detection of SARS-CoV-2. A specimen is considered positive for 2019-nCoV if both assays are positive, whereas if only one assay resulted positive the result is inconclusive and it should be retested.Centers of Disease Control and Prevention, 2020a, 2020bNMDC panelc2RdRp, NThis panel contains two monoplex assays designed for specific detection of SARS-CoV-2. A specimen is considered positive for 2019-nCoV if both assays are positive.National Microbiology Data Center, 2020HKU paneld2RdRp, NThe N gene RT-PCR is recommended as a screening assay and the Orf1b assay as a confirmatory one (the N gene assay is about 10 times more sensitive than the ORF-1b gene assay in detecting positive clinical specimens).Chu et al., 2020; The University of Hong Kong, 2020.QIAstat-Dx Panele2RdRp, EThis panel contains a Duplex assay in the same optical channel to report an additive performance (including CT and endpoint fluorescence values) of the two PCR assays and it discriminates SARS-CoV-2 detection from more than other 20 viruses and bacteria responsible for upper respiratory infections including other endemic coronavirusesQIAstat-Dx Respiratoyry SARS-CoV-2 Panel, 2020RdRp: RNA-dependent RNA polymerase gene (inside the Orf1ab polyprotein gene), E: envelop gene, N: Nucleocaspide gene.This panel was originally proposed by the Charité-Universitätsmedizin Berlin Institute of Virology (Corman et al., 2020), and then endorsed by the WHO (World Helath Organization, 2020b).Initial CDC panel contained one additional assay (N3) designed for universal detection of SARS-like coronaviruses (Centers of Disease Control and Prevention, 2020a) but has been removed due to inconclusive results based on low performance (Centers of Disease Control and Prevention, 2020b).National Microbiology Data Center (NMDC) works in collaboration with National Institute for Viral Disease Control and Prevention under Chinese Center for Disease Control and Prevention.The School of Public Health of the University of Hong Kong (HKU) developed the assay (Chu et al., 2020) and it was later established as official protocol under the WHO Network Laboratories (The University of Hong Kong, 2020).QIAstat-Dx Panel is a syndromic Point-of-Care system incorporating sample purification and real time RT-qPCR amplification for more than 20 viral and bacterial respiratory pathogens, including SARS-CoV-2.WHO and HKU assays are reactive with coronaviruses under the subgenus Sarbecovirus that includes 2019-nCoV, SARS-CoV and bat SARS-like coronaviruses. Since SARS was eliminated in humans since 2004, individuals with samples that are positive in these RT-PCR assays should be infected by the 2019-nCoV or its related animal coronaviruses.

Lifting the lid on toilet plume aerosol: A literature review with suggestions for future research

Bioaerosols, old news, but relevant today.

“The potential for airborne transmission of sewage-related infectious disease was demonstrated by Horrocks over 100 years ago when he cultured airborne microorganisms from sewage drain systems and also detected airborne transport from one hospital building to another via the sewer drains. Similar results were seen by others including Andrewes.”

Today this bears repeating: Bioaerosols ARE a cause of infectious disease transmission.

This literature review by Dr. Deborah Hirst, Ph.D. and her colleagues summarizes one area of research in bioaerosols and infectious disease transmission.

ABSTRACT:

Background

The potential risks associated with “toilet plume” aerosols produced by flush toilets is a subject of continuing study. This review examines the evidence regarding toilet plume bioaerosol generation and infectious disease transmission.

Methods

The peer-reviewed scientific literature was searched to identify articles related to aerosol production during toilet flushing, as well as epidemiologic studies examining the potential role of toilets in infectious disease outbreaks.

Results

The studies demonstrate that potentially infectious aerosols may be produced in substantial quantities during flushing. Aerosolization can continue through multiple flushes to expose subsequent toilet users. Some of the aerosols desiccate to become droplet nuclei and remain adrift in the air currents. However, no studies have yet clearly demonstrated or refuted toilet plume-related disease transmission, and the significance of the risk remains largely uncharacterized.

Conclusion

Research suggests that toilet plume could play a contributory role in the transmission of infectious diseases. Additional research in multiple areas is warranted to assess the risks posed by toilet plume, especially within health care facilities.

Am J Infect Control. 2013 Mar; 41(3): 254–258.
Published online 2012 Oct 5. 
doi: 10.1016/j.ajic.2012.04.330

Coupling Polar Adhesion with Traction, Spring, and Torque Forces Allows High-Speed Helical Migration of the Protozoan Parasite Toxoplasma

Parasitic motility has been a fascination of mine as my thesis characterized microtubule-based motor proteins in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT) and the animal wasting disease na’gana. Here is a video of that motion.

The American Chemical Society has just released a publication characterizing biophysical characteristics involved in the gliding motion of the parasite Toxoplasma gondii. Plasmodium parasites (the causative agents of malaria) and Toxoplasma parasites use the same cellular machinery to achieve this gliding. The authors use sophisticated microscopy techniques (reflection interference microscopy and expansion microscopy) to build their observational model describing cytoskeletal changes that occur to allow the characteristic motion of left-handed gliding. The authors summarize:

“We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.”

AUTHORS:

Georgios Pavlou, Bastien Touquet, Luis Vigetti, Patricia Renesto, Alexandre Bougdour, Delphine Debarre, Martial Balland, Isabelle Tardieux*

ABSTRACT:

Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion–de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.

ACS Nano 2020, XXXX, XXX, XXX-XXX
Online Publication Date: May 20, 2020
https://doi.org/10.1021/acsnano.0c01893

Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways

On July 9, 1878, Carlos Ribiero Justiniano Chagas was born on a coffee farm in Brazil. Carlos would go on to become a physician and work in the tropical medicine field and coordinate the response role in combating the 1918 influenza pandemic in Brazil.

Fast forward to present day, Chagas disease, a disease he first characterized in 1909, caused by the Trypanosoma cruzi parasite now afflicts 6 to 8 million people worldwide. While endemic to Central and South America, many countries in Europe, the United States, Japan, and Australia also have Chagas disease burden including an estimated 100,000 cases in Europe and 300,000 in the United States.

Just last month, Mr. Jacob M. Wozniak et al. published this peer-reviewed research article on the study of proteins and protein modifications upregulated in mice infected with Trypanosoma cruzi. The authors summarize:
“For the first time, this study provides insight into the in vivo host signaling responses to T. cruzi in the heart, uncovering pathways that can be validated for contributions to disease and suitability as drug targets…
This study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.”

AUTHORS: Jacob M. Wozniak, Tatiana Araújo Silva, Diane Thomas, Jair L. Siqueira-Neto, James H. McKerrow, David J. Gonzalez, and Claudia M. Calvet 

AUTHOR SUMMARY: Chagas disease is a significant human health concern as it can cause severe cardiomyopathies in chronically infected patients. Due to the high morbidity associated with Chagasic cardiomyopathies, it is vital to investigate new treatment options. In this study, we use state-of-the-art techniques to interrogate the host signaling events induced by chronic Chagas disease in the primary affected organ, the heart. We identify proteins and phospho-sites significantly altered upon infection, predict activated and inhibited kinases, and link our findings to known drug targets. For the first time, this study provides insight into the in vivo host signaling responses to T. cruzi in the heart, uncovering pathways that can be validated for contributions to disease and suitability as drug targets.

ABSTRACT: Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.

AUTHOR AFFILIATIONS:

Jacob M. Wozniak

ROLES Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Writing – original draft, Writing – review & editing

AFFILIATIONS Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America, Department of Pharmacology; University of California San Diego; La Jolla, CA, United States of America

http://orcid.org/0000-0002-2453-0861

Tatiana Araújo Silva

ROLES Investigation

AFFILIATION Cellular Ultrastructure Laboratory; Oswaldo Cruz Institute, FIOCRUZ; Rio de Janeiro, RJ, Brazil

Diane Thomas

ROLES Investigation, Writing – review & editing

AFFILIATION Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America

Jair L. Siqueira-Neto

ROLES Resources, Writing – review & editing

AFFILIATION Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America

http://orcid.org/0000-0001-9574-8174

James H. McKerrow

ROLES Resources

AFFILIATION Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America

David J. Gonzalez

ROLES Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing

* E-mail: djgonzalez@ucsd.edu (DJG); claudiacalvet@gmail.com (CMC)

AFFILIATIONS Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America, Department of Pharmacology; University of California San Diego; La Jolla, CA, United States of America

Claudia M. Calvet

ROLES Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – review & editing

* E-mail: djgonzalez@ucsd.edu (DJG); claudiacalvet@gmail.com (CMC)

AFFILIATIONS Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America, Cellular Ultrastructure Laboratory; Oswaldo Cruz Institute, FIOCRUZ; Rio de Janeiro, RJ, Brazil

http://orcid.org/0000-0003-1275-1226

Competing Interests

The authors have declared that no competing interests exist.

Age, sex and disease-specific associations between resting heart rate and cardiovascular mortality in the UK BIOBANK

Dr. Zahra Raisi-Estabragh and colleagues summarize a very LARGE cohort study of over 500,000 individuals in the UK Biobank. Entering my 40s has caused me to consider careful monitoring of many health indicators including my resting heart rate. The authors concluded that with each increase in 10 beats per minute (bpm) there was a 22% and 17%, in males and females respectively, rise in all cause cardiovascular disease. Additional metrics for cancer mortality were also measured. A link to the full article published last week in the Public Library of Science (PLOS ONE) open-access journal can be accessed here. Another interesting but unrelated PLOS ONE article (and blast from the past of 2011) can be accessed here.

The authors conclude:

”RHR is an independent predictor of mortality, with variation by sex, age, and disease. Ischaemic disease appeared a more important driver of this relationship in men, and associations were more pronounced at younger ages.”

AUTHORS: Zahra Raisi-Estabragh, Jackie Cooper, Rebekah Judge, Mohammed Y. Khanji, Patricia B. Munroe, Cyrus Cooper, Nicholas C. Harvey, Steffen E. Petersen 

ABSTRACT:

OBJECTIVE:

To define the sex, age, and disease-specific associations of resting heart rate (RHR) with cardiovascular and mortality outcomes in 502,534 individuals from the UK Biobank over 7–12 years of prospective follow-up.

METHODS:

The main outcomes were all-cause, cardiovascular, and ischaemic heart disease mortality. Additional outcomes included incident acute myocardial infarction (AMI), fatal AMI, and cancer mortality. We considered a wide range of confounders and the effects of competing hazards. Results are reported as hazard ratios (HR) for all-cause mortality and sub-distribution hazard ratios (SHR) for other outcomes with corresponding 95% confidence intervals (CI) per 10bpm increment of RHR.

RESULTS:

In men, for every 10 bpm increase of RHR there was 22% (HR 1.22, CI 1.20 to 1.24, p = 3×10−123) greater hazard of all-cause and 17% (SHR 1.17, CI 1.13 to 1.21, p = 5.6×10−18) greater hazard of cardiovascular mortality; for women, corresponding figures were 19% (HR 1.19, CI 1.16 to 1.22, p = 8.9×10−45) and 14% (SHR 1.14, CI 1.07 to 1.22, p = 0.00008). Associations between RHR and ischaemic outcomes were of greater magnitude amongst men than women, but with similar magnitude of association for non-cardiovascular cancer mortality [men (SHR 1.18, CI 1.15–1.21, p = 5.2×10−46); women 15% (SHR 1.15, CI 1.11–1.18, p = 3.1×10−18)]. Associations with all-cause, incident AMI, and cancer mortality were of greater magnitude at younger than older ages.

CONCLUSIONS:

RHR is an independent predictor of mortality, with variation by sex, age, and disease. Ischaemic disease appeared a more important driver of this relationship in men, and associations were more pronounced at younger ages.

ABOUT THE AUTHORS:

Zahra Raisi-Estabragh

ROLES Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

AFFILIATIONS William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom, Barts Heart Centre, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom

http://orcid.org/0000-0002-7757-5465

Jackie Cooper

ROLES Data curation, Formal analysis, Methodology, Software, Writing – review & editing

AFFILIATIONS William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom, Barts Heart Centre, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom

Rebekah Judge

ROLES Writing – original draft, Writing – review & editing

AFFILIATION Imperial College Healthcare Trust, St Mary’s Hospital, London, United Kingdom

http://orcid.org/0000-0003-3288-8297

Mohammed Y. Khanji

ROLES Writing – review & editing

AFFILIATIONS William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom, Barts Heart Centre, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom

Patricia B. Munroe

ROLES Writing – review & editing

AFFILIATION William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom

Cyrus Cooper

ROLES Writing – review & editing

AFFILIATIONS MRC Lifecourse Epidemiology Unit (MRCLEU), Southampton, United Kingdom, NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom, NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom

Nicholas C. Harvey

ROLES Conceptualization, Methodology, Supervision, Writing – original draft, Writing – review & editing

AFFILIATIONS MRC Lifecourse Epidemiology Unit (MRCLEU), Southampton, United Kingdom, NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom

Steffen E. Petersen

ROLES Conceptualization, Methodology, Supervision, Writing – original draft, Writing – review & editing

* E-mail: s.e.petersen@qmul.ac.uk

AFFILIATIONS William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom, Barts Heart Centre, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom

http://orcid.org/0000-0003-4622-5160

Competing Interests

I have read the journal's policy and the authors of this manuscript have the following competing interests: SEP acts as a paid consultant to Circle Cardiovascular Imaging Inc., Calgary, Canada and Servier. NCH has received consultancy, lecture fees, and honoraria from Alliance for Better Bone Health, Amgen, MSD, Eli Lilly, Servier, Shire, Radius Health, UCB, Consilient Healthcare, Kyowa Kirin and Internis Pharma. The remaining authors have nothing to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Airway mir-155 responses are associated with TH1 cytokine polarization in young children with viral respiratory infections

Many of you know I have a love of RNA and the role of microRNAs in immune modulation. Some of you don’t even know what this means and don’t know that is a passion of mine.

I have had my son fall ill from RSV twice due to unnecessary exposure from children that presented as ill but with what seemed by their caregivers as a harmless and minor “cold.” Please read and beware.

The PLOS link is here.

ABSTRACT:
Background

MicroRNAs (miRs) control gene expression and the development of the immune system and antiviral responses. MiR-155 is an evolutionarily-conserved molecule consistently induced during viral infections in different cell systems. Notably, there is still an unresolved paradox for the role of miR-155 during viral respiratory infections. Despite being essential for host antiviral TH1 immunity, miR-155 may also contribute to respiratory disease by enhancing allergic TH2 responses and NFkB-mediated inflammation. The central goal of this study was to define how airway miR-155 production is related to TH1, TH2, and pro-inflammatory cytokine responses during naturally occurring viral respiratory infections in young children.

Methods

Normalized nasal airway levels of miR-155 and nasal protein levels of IFN-γ, TNF-α, IL-1β, IL-13, IL-4 were quantified in young children (≤2 years) hospitalized with viral respiratory infections and uninfected controls. These data were linked to individual characteristics and respiratory disease parameters.

Results

A total of 151 subjects were included. Increased miR-155 levels were observed in nasal samples from patients with rhinovirus, RSV and all respiratory viruses analyzed. High miR-155 levels were strongly associated with high IFN-γ production, increased airway TH1 cytokine polarization (IFN-γ/IL-4 ratios) and increased pro-inflammatory responses. High airway miR-155 levels were linked to decreased respiratory disease severity in individuals with high airway TH1 antiviral responses.

Conclusions

The airway secretion of miR-155 during viral respiratory infections in young children is associated with enhanced antiviral immunity (TH1 polarization). Further studies are needed to define additional physiological roles of miR-155 in the respiratory tract of human infants and young children during health and disease.

AUTHORS:

Maria Arroyo, Kyle Salka, Elizabeth Chorvinsky, Xilei Xuchen, Karima Abutaleb, Geovanny F. Perez, Jered Weinstock, Susana Gaviria, Maria J. Gutierrez, Gustavo Nino 

Published: May 22, 2020

https://doi.org/10.1371/journal.pone.0233352

Time series modelling to forecast the confirmed and recovered cases of COVID-19

The authors describe the forecasting of both confirmed and recovered cases of SARS-CoV-2 through April of 2020. Figure 6 below illustrates the recovered cases from April 21st to April 30, 2020.

Maleki et al. conclude:

”There exist many situations in the real world that the assumption of symmetric distribution of the error terms is not satisfactory. In our methodology, we considered autoregressive time series models based on the two–piece scale mixturenormal (TP–SMN) distributions. The results indicated that the proposed method performed well in forecasting confirmed and recovered COVID-19 cases in the world. Using model selection criteria, the proposed models were also more reasonable than the standard Gaussian autoregressive time series model which is the simplest member of our proposed models. For future works, we suggest that the researchers apply cyclostationary, almost cyclostationary, and simple processes based on the TP–SMN distributions, instead of stationary processes.”

AUTHORS: Mohsen Maleki, Mohammad Reza, Mahmoudi Darren Wraith, Kim-Hung Pho

AUTHOR AFFILIATIONS:
a Department of Statistics, University of Isfahan, Isfahan, Iran
b Institute of Research and Development, Duy Tan University, Da Nang, 550000, Vietnam
c Department of Statistics, Faculty of Science, Fasa University, Fasa, Fars, Iran
d Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Queensland, Australia
e Fractional Calculus, Optimization and Algebra Research Group, Faculty of Mathematics and Statistics, Ton Duc Thang University, Ho Chi Minh City, Vietnam

CORRESPONDING AUTHORS:
E-mail addresses:
m.maleki.stat@gmail.com (M. Maleki),
mohammadrezamahmoudi@duytan.edu or vnmahmoudi.m.r@fasau.ac.ir (M.R. Mahmoudi)
d.wraith@qut.edu.au (D. Wraith)
phokimhung@tdtu.edu.vn (K.-H. Pho)

ABSTRACT:
Coronaviruses are enveloped RNA viruses from the Coronaviridae family affecting neurological, gastrointestinal, hepatic, and respiratory systems. In late 2019 a new member of this family belonging to the Betacoronavirus genera (referred to as COVID-19) originated and spread quickly across the world calling for strict containmentplans and policies. In most countries in the world, the outbreak of the disease has been serious and the number of confirmed COVID-19 cases has increased daily, while, fortunately the recovered COVID-19 cases have also increased. Clearly, forecasting the “confirmed” and “recovered” COVID-19 cases helps planning to control the disease and plan for utilization of health care resources. Time series models based on statistical methodology are useful to model time-indexed data and for forecasting. Autoregressive time series models based on two-piece scale mixture normal distributions, called TP–SMN–AR models, is a flexible family of models involving many classical symmetric/asymmetric and light/heavy tailed autoregressive models. In this paper, we use this family of models to analyze the real world time series data of confirmed and recovered COVID-19 cases.

Ref.https://doi.org/10.1016/j.tmaid.2020.101742Received 9 March 2020; Received in revised form 7 May 2020; Accepted 9 May 2020∗

Travel Medicine and Infectious Disease xxx (xxxx) xxxx1477-8939/ © 2020 Published by Elsevier Ltd.
Please cite this article as: Mohsen Maleki, et al., Travel Medicine and Infectious Disease,

https://doi.org/10.1016/j.tmaid.2020.101742

On setting expectations for a SARS-CoV-2 Vaccine

I have been fascinated with infectious diseases for 36 years and pandemics specifically since my introductory Pathobiology course at University. I recall interviewing my paternal grandmother and other friends her age that lived through the 1918 pandemic and outbreaks in the Belgian Congo.

Fast forward 20 years and today, as predicted nearly to the year by my pathobiology instructors, we are in the throes of another pandemic. Dr. David H Canaday, MD and Dr. Stefan Gravenstein, MD, MPH have written about the international collaborations to develop a vaccine for SARS-CoV-2. The full text article can be accessed here.

AUTHORS: David H Canaday, MD, Stefan Gravenstein, MD, MPH

David H. Canaday, MD
Division of Infectious Diseases
Case Western Reserve University Division of Infectious Diseases
Geriatric Research, Education and Clinical Center (GRECC)
Cleveland Veterans Administration Medical Center Cleveland, OH, U.S.A.

Stefan Gravenstein, MD, MPH
Alpert Medical School and School of Public Health,
Brown University
Providence Veteran Administration Medical Center Providence, RI, U.S.A.

ABSTRACT: The global coronavirus pandemic is unlike any other since 1918. A century of dramatic medical advances has produced a public expectation that the medical field will rapidly provide solutions to restore normalcy. In under 6 months, since SARS-CoV-2 was identified, the massive international effort to develop a SARS-CoV-2 vaccine has generated more than 140 vaccines in different stages of development with 9 already recruiting into clinical trials posted on clinicaltrials.gov. The long-term strategy to handle COVID-19 will almost certainly rely on vaccines. But, what type of protection can we realistically expect to achieve from vaccines and when?

Published by Oxford University Press for the Infectious Diseases Society of America 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US.

This work is written by (a) US Government employee(s) and is in the public domain in the US.

DOI: Clinical Infectious Diseases, ciaa726, 
https://doi.org/10.1093/cid/ciaa726

PUBLISHED:

04 June 2020

The impact of ethnicity on clinical outcomes in COVID-19: A systematic review

This is the FIRST review analysis on the outcomes of ethnicity on the clinical outcomes of SARS-CoV-2 infection. The authors concluded that:

“Black Asian Minority or Ethnic (BAME) individuals are at an increased risk of acquiring SARS-CoV-2 infection compared to White individuals and also worse clinical outcomes from COVID-19. Further work on the role of ethnicity in the current pandemic is of urgent public health importance.”

AUTHORS: Daniel Pana, b,1 , Shirley Szec,1 , Jatinder S. Minhas c , Mansoor N. Bangash d,e , Nilesh Pareek f, Pip Divall g , Caroline ML. Williams a,b , Marco R. Oggionih , Iain B. Squirec , Laura B. Nellumsi , Wasim Hanif j, Kamlesh Khunti k , Manish Pareek a,b, * a

AUTHOR AFFILIATIONS:
a Department of Respiratory Sciences, University of Leicester, United Kingdom
b Department of Infection and HIV Medicine, Leicester Royal Infirmary, University Hospitals Leicester NHS Trust, United Kingdom
c Department of Cardiovascular Sciences, University of Leicester, United Kingdom
d Department of Intensive Care, University Hospitals Birmingham NHS Foundation Trust, United Kingdom
e Institute of Clinical Sciences, University of Birmingham, United Kingdom
f School of Cardiovascular Medicine and Sciences, King’s BHF Centre of Excellence, London, United Kingdom
g University Hospitals of Leicester, Education Centre Library, Glenfield Hospital and Leicester Royal Infirmary, United Kingdom
h Department of Genetics and Genome Biology, University of Leicester, United Kingdom
i Faculty of Medicine and Health Sciences, University of Nottingham, United Kingdom
j Department of Diabetes and Endocrinology, University Hospitals Birmingham NHS Foundation Trust, United Kingdom
k Leicester Diabetes Centre, University of Leicester, United Kingdom

ABSTRACT:
Background: The relationship between ethnicity and COVID-19 is uncertain. We performed a systematic review to assess whether ethnicity has been reported in patients with COVID-19 and its relation to clinical outcomes.

Methods: We searched EMBASE, MEDLINE, Cochrane Library and PROSPERO for English-language citations on ethnicity and COVID-19 (1st December 2019-15th May 2020). We also reviewed: COVID-19 articles in NEJM, Lancet, BMJ, JAMA, clinical trial protocols, grey literature, surveillance data and preprint articles on COVID-19 in MedRxiv to evaluate if the association between ethnicity and clinical outcomes were reported and what they showed. PROSPERO:180654.

Findings: Of 207 articles in the database search, five reported ethnicity; two reported no association between ethnicity and mortality. Of 690 articles identified from medical journals, 12 reported ethnicity; three reported no association between ethnicity and mortality. Of 209 preprints, 34 reported ethnicity 13 found Black, Asian and Minority Ethnic (BAME) individuals had an increased risk of infection with SARS-CoV-2 and 12 reported worse clinical outcomes, including ITU admission and mortality, in BAME patients compared to White patients. Of 12 grey literature reports, seven with original data reported poorer clinical outcomes in BAME groups compared to White groups.

Interpretation: Data on ethnicity in patients with COVID-19 in the published medical literature remains limited. However, emerging data from the grey literature and preprint articles suggest BAME individuals are at an increased risk of acquiring SARS-CoV-2 infection compared to White individuals and also worse clinical outcomes from COVID-19. Further work on the role of ethnicity in the current pandemic is of urgent public health importance.

Funding: NIHR © 2020

Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

Please cite this article as: D. Pan et al., The impact of ethnicity on clinical outcomes in COVID-19: A systematic review, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100404

Introductions and early spread of SARS-CoV-2 in the New York City area

This publication from Science shares the rapid rise in SARS-CoV-2 cases in March of 2020 in New York City. Figure one above shows the number of patients with positive molecular tests for SARS-CoV-2 up to March 31 2020 compared to the daily number of patients with influenza A and influenza B tests in the 2019-2020 season.

AUTHORS: Ana S. Gonzalez-Reiche1,*,Matthew M. Hernandez2,3,*,Mitchell J. Sullivan1, Brianne Ciferri1, Hala Alshammary2, Ajay Obla1, Shelcie Fabre4, Giulio Kleiner2, Jose Polanco1,2, Zenab Khan1, Bremy Alburquerque1,3, Adriana van de Guchte1, Jayeeta Dutta1, Nancy Francoeur1, Betsaida Salom Melo1,5, Irina Oussenko1,5, Gintaras Deikus1,5, Juan Soto1,5, Emilia Mia Sordillo3,†,‡, Viviana Simon2,5,6,†,‡, Harm van Bakel1,8,†,‡

1. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

2. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

3. The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

4. Clinical Microbiology Laboratory, Department of Pathology, Molecular, and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

5. Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

6. The Mount Sinai Data Office, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

7. Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

8. Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

9. Sema4, a Mount Sinai venture, Stamford, CT 06902, USA.

10. The Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

11. The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

12. Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

CORRESPONDING AUTHOR:
illo@mssm.edu (E.M.S.)

↵* These authors contributed equally to this work.

↵‡ These authors contributed equally to this work.

↵* These authors contributed equally to this work.

↵‡ These authors contributed equally to this work.

ABSTRACT: New York City (NYC) has emerged as one of the epicenters of the current SARS-CoV-2 pandemic. To identify the early transmission events underlying the rapid spread of the virus in the NYC metropolitan area, we sequenced the virus causing COVID-19 in patients seeking care at the Mount Sinai Health System. Phylogenetic analysis of 84 distinct SARS-CoV2 genomes indicates multiple, independent but isolated introductions mainly from Europe and other parts of the United States. Moreover, we find evidence for community transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different neighborhoods of the city.

Science  29 May 2020:
eabc1917
DOI: 10.1126/science.abc1917

FIGURE 2:

Rapid repurposing of drugs for COVID-19

This is an excerpt from the Science Magazine perspectives article in the May 22, 2020 issue.

“In late fall 2019, a novel acute respiratory disease, called coronavirus disease 2019 (COVID-19) emerged in Wuhan, China. COVID-19 is caused by severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) (1, 2). COVID-19 has been declared a pandemic by the World Health Organization and continues to spread across the globe. Most patients recover within 1 to 3 weeks. However, a small proportion (∼5%) develop severe illness that can progress to acute respiratory distress syndrome (ARDS), which can lead to death. Currently, only supportive care is available; patients would greatly benefit from the availability of direct therapeutic approaches. One approach to identifying therapeutics is to repurpose approved drugs developed for other uses, which takes advantage of existing detailed information on human pharmacology and toxicology to enable rapid clinical trials and regulatory review.

The coronaviruses are single-stranded RNA viruses that infect vertebrates and move between different host species (3). With the emergence of SARS-CoV-2, there are now seven coronaviruses that are known to infect humans. Four of them (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) are responsible for ∼30% of cases of the common cold in humans. Two of them caused recent epidemics that had considerable associated mortality: SARS-CoV-1, which emerged in 2002–2003 and causes ∼10% mortality, and Middle East respiratory syndrome coronavirus (MERS-CoV), which emerged in 2012, is still active, and causes ∼35% mortality. Both epidemics affected a relatively small number of patients compared with COVID-19, which is more transmissible for several reasons, including asymptomatic carriers, long latency period, and high infectivity. Before COVID-19, only SARS-CoV-1 and MERS-CoV caused severe disease. Therefore, coronaviral drug discovery has been a small effort relative to that for other viral diseases such as influenza. Given the rapid spread of COVID-19 and its relatively high mortality, filling the gap for coronavirus-specific drugs is urgent.

The coronavirus life cycle (see the figure) involves a number of potentially targetable steps, including endocytic entry into host cells [involving angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2)], RNA replication and transcription [involving helicase and RNA-dependent RNA polymerase (RdRp)], translation and proteolytic processing of viral proteins (involving chymotrypsin-like and papain-like proteases), virion assembly, and release of new viruses through the exocytic systems (4). In addition to virally encoded targets, numerous host targets are essential for viral replication and disease progression (3).

The cellular receptor for SARS-CoV-2 is ACE2 (5). Recombinant human ACE2 (rhACE2, or APN01) is currently under development as a treatment for acute lung injury and pulmonary arterial hypertension and has proven well tolerated in a phase 1 trial in healthy volunteers. rhACE2 has been shown to significantly reduce viral entry into human cell–derived organoids (6), presumably by acting as a decoy for virus binding. This has lent support to the clinical trials that are investigating blockade of viral entry with APN01 for COVID-19 patients. Successful viral entry requires proteolytic processing of the viral coat spike glycoprotein (S), which can be carried out by TMPRSS2 (7). The TMPRSS2 inhibitor camostat (7) is approved in Japan for the treatment of chronic pancreatitis and postoperative gastric reflux and is generally well tolerated, although rare serious side effects have been reported. Both camostat and the related agent nafamostat (8) block SARS-CoV-2 replication in TMPRSS2-expressing human cells. Camostat has been shown to block infection with SARS-CoV-2 in a mouse model. Therefore, there is a strong rationale to support clinical trials with these drugs for COVID-19, which have already been initiated in the Netherlands and Germany.

Coronaviruses use the endolysosomal pathway to enter the cell before uncoating. Chloroquine (CQ) and hydroxychloroquine (HCQ) are antimalarial drugs that affect endosomal function and block autophagosome-lysosome fusion (9). Both drugs have been shown to inhibit SARS-CoV-2 replication in cellular models (8, 10). Azithromycin (AZ), a widely used broad-spectrum antibiotic, also blocks autophagosome clearance in human cells (11) and replication of the Zika virus and influenza virus in human cells in vitro (12). Preliminary results from a small randomized trial of HCQ in COVID-19 patients report a reduction in time to clinical resolution (13). A small open-label trial has demonstrated increased reduction in viral load for COVID-19 patients receiving the combination of HCQ and AZ relative to HCQ alone, although this study has been heavily criticized because of post hoc removal of several subjects from the study analysis (14). These hypothesis-generating studies have justified emergency approval of their use for COVID-19 in the United States, where they are both being widely used.

However, both HCQ and AZ have potential cardiac toxicity (QT prolongation, which can lead to fatal arrhythmia), and HCQ additionally has the potential for negative effects on the eye. Understanding risk-benefit ratios is paramount if these drugs are to become a standard of care for COVID-19. Several post hoc analyses carried out in the United States and Europe suggest modest benefit, at best, from HCQ monotherapy for COVID-19 patients; one large post hoc analysis among U.S. veterans suggests that there is harm to patients from HCQ. Given the mechanistic rationale but lack of well-designed clinical studies and potential for drug-induced toxicity, there is a key need for controlled, randomized trials to test the efficacy and safety of these drugs for COVID-19 patients.

After uncoating, the viral genomic RNA is used for cap-dependent translation to produce two polypeptides, which are then autoproteolytically processed to produce several viral proteins, including RdRp and two proteases. Although the proteases might seem attractive targets given the number of viral protease inhibitors previously developed for HIV and other viruses, they are only distantly related to other viral proteases. The combination of the HIV protease inhibitors lopinavir and ritonavir (15) proved clinically ineffective for COVID-19 patients, as had previously been the case for the same combination in SARS-CoV-1 disease. Therefore, further repurposing with this class of drugs is poorly justified—although there are other protease inhibitors in early-stage drug discovery that are directed to the coronavirus proteases.

Production of the replication complex proteins, including the helicase and RdRp, allows for genomic replication of the virus and for production of subgenomic RNAs, which are also translated to produce structural and coat proteins. The helicase is theoretically an attractive target, but it is divergent from other viral helicases, and there is no evidence that the herpes simplex virus helicase inhibitors amenamevir or pretelivir are effective against coronaviruses.

RdRp carries out both replication and transcription of the viral RNA, making it a clear target for blocking the viral life cycle. Because RdRp is a critical protein for many viruses, a number of broad-spectrum RdRp inhibitors are either approved or in clinical trials, including remdesivir and favipiravir. Remdesivir was initially developed to treat the flaviviruses that cause Ebola and Marburg diseases and has proven safe in trials during the past two Ebola epidemics. However, it is less effective for Ebola than antibody-based treatments that prevent the virus from entering human cells. Remdesivir was subsequently shown to be active against both SARS-CoV-1 and MERS-CoV in animal models. Favipiravir was developed for influenza and approved in Japan in 2014, specifically for new pandemic influenza outbreaks. Both remdesivir and favipiravir are active against SARS-CoV-2 in human cells in vitro (7). Remdesivir has been rapidly advanced into several clinical trials for COVID-19, and early informal data being released from those trials suggest that remdesivir is effective, but such datasets need to be used cautiously for generalizing the understanding of either safety or efficacy. Further randomized, controlled trials with RdRp inhibitors are justified and needed.

The best justified drugs for repurposing to treat COVID-19 patients are the host-factor–targeted drugs HCQ, AZ, and camostat and nafamostat and the viral RdRp–targeted drugs remdesivir and favipiravir. A number of other drugs are also being considered, although with less supporting evidence (see supplementary materials). Additionally, phenotypic screening approaches are being developed on the basis of either viral entry or replication that could be used to survey approved drugs and drug candidates much more widely. Both of these approaches may widen the available classes of drugs for consideration.

The key issue with any of these potential treatments is to balance the oppositional needs of making treatment decisions for individual patients during epidemic peaks on the basis of clinical studies that involve small numbers of patients with ensuring that well-designed, randomized clinical trials are carried out rapidly to provide proof that they are safe and efficacious. COVID-19 is expected to be active permanently, and several seasons of disease peaks are likely before herd (population) immunity is established. The difficulty is to coordinate rapid hypothesis-generating studies during this first peak to justify a smaller number of well-controlled large trials to be executed in later peaks to provide the data needed for approval of drugs for COVID-19. Researchers, ethics boards, and regulators are accustomed to developing trial plans over months, not weeks—a time frame that is not afforded during this emergent situation. It is necessary for all involved to work faster and more efficiently and then position the well-justified drugs for registration-enabling trials during the next peak.”

Excess mortality in men and women in Massachusetts during the COVID-19 pandemic.

The Lancet has published a correspondence about the research of Dr. Nancy Krieger, PhD and her colleagues at Harvard University. The piece comes after many states have grappled with statistics on SARS-CoV-2 -related deaths in their own states as well as the various sex-differences between females and males that succumb to SARS-CoV-2. The comparative data Dr. Krieger shares from 2015-2019 in contrast to 2020 are staggering as seen here.

AUTHORS: Krieger N, Chen JT, Waterman PD

CITATION: Lancet 2020; published online May 27.
http://dx.doi.org/10.1016/S0140-6736(20)31234-4.

CORRESPONDING AUTHOR:
Nancy Krieger, PhD
Professor of Social Epidemiology
American Cancer Society Clinical Research Professor
Department of Social and Behavioral Sciences
Harvard T.H. Chan School of Public Health
677 Huntington Avenue Boston, MA 02115
Email: nkrieger@hsph.harvard.edu

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial

Results published today in the Lancet describe a study of 108 individuals given an intramuscular (IM) dose of the Adenovirus 5 vaccine that recombinantly-expresses the spike glycoprotein of the SARS-CoV-2 coronavirus. This is the FIRST IN HUMAN safety and tolerability vaccine publication to date. At least 89% of the dose-challenged participants had no underlying condition. The researchers report that a total of 9% (10 individuals) had a Grade 3 reaction. Fifty percent of low to medium-dosed groups and 75% of the high-dose subjects had neutralizing antibodies to SARS-CoV-2, 28 days post IM dose. The authors summarize:

“The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation.”

AUTHORS: Feng-Cai Zhu*, Yu- Hua Li*, Xu-Hua Guan, Li-Hua Hou, Wen-Juan Wang, Jing-Xin Li, Shi-Po Wu, Bu-Sen Wang, Zhao Wang, Lei Wang, Si-Yue Jia, Hu-Dachuan Jiang, Ling Wang, Tao Jiang, Yi Hu, Jin-Bo Gou, Sha-Bei Xu, Jun-Jie Xu, Xue-Wen Wang, Wei Wang, Wei Chen

CORRESPONDENCE:
Professor Wei Chen, PhD, Beijing Institute of Biotechnology, Beijing 100071, China
Email: cw0226@foxmail.com
Professor Wei Wang, PhD, Clinical Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 43003
Email: wwang@vip.126.com

BACKGROUND: A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. Methods We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled and allocated to one of three dose groups (5×10¹⁰, 1×10¹¹, and 1·5×10¹¹ viral particles) to receive an intramuscular injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127.

FINDINGS: Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination.

INTERPRETATION: The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation.