The Northern Paraguai Belt, at the SE border of the Amazonian Craton, central Brazil, has been interpreted as a Brasiliano–Pan-African (c. 650–600 Ma) belt with a foreland basin, recording collisional polyphase tectonism and greenschist-facies metamorphism extending from the late Precambrian to the Cambrian–Ordovician. New structural investigations indicate that the older metasedimentary rocks of the Cuiabá Group represent a Tonian–Cryogenian basement assemblage deformed in two contemporaneous fault-bounded structural sub-domains of wrench-dominated (rake <10°) and contraction-dominated (rake ~30–40°) sinistral transpression, with tectonic vergence towards the SE. The younger late Cryogenian to early Cambrian sedimentary rocks lying to the NW of the Cuiabá Group are non-metamorphic and display only pervasive brittle transtension characterized by normal-oblique faults, fractures and forced drag folds with no consistent vergence pattern. Our analyses suggest that an unconformity separates the metasedimentary Cuiabá Group basement of the Northern Paraguai Belt from the unmetamorphosed sedimentary cover. It is proposed that the latter units were deposited during a post-glacial marine transgression (after c. 635 Ma) in a post-collisional basin. The Paraguai Belt basement and its post-collisional sedimentary cover share a number of significant geological similarities with sequences in the Bassarides Belt and Taoudéni Basin in the SW portion of the West African Craton.

The Tabaquito batholith (Frontal Cordillera, western Argentina), is mainly composed of shallowly emplaced granodiorite to minor monzogranite with abundant mafic microgranular enclaves. New sensitive high-resolution ion microprobe U–Pb zircon ages of c. 337 Ma (biotite granodiorite) and c. 284 Ma (mafic dyke) along with previously published geochronological data suggest that a long-lived magmatic system formed through at least two magmatic pulses at c. 337 and c. 322 Ma with later superimposition of Permian magmatism. The Tabaquito granitoids are metaluminous, calc-alkalic and magnesian with I-type affinity. Elevated Th/Nb, Y/Nb and La/Nb ratios along with negative Nb–Ta and positive Pb anomalies are consistent with a continental arc setting. Hf, Nd and Sr isotopic composition of the Tabaquito granitoids suggests that their source could result from mixing of an old felsic crustal component and a juvenile mafic to intermediate component. New geochronological and geochemical data together with published data reveal a continuous arc setting from the Carboniferous to the Permian in Argentina, and important magmatic compositional variations through time and space controlled by episodic fluctuations in the subduction angle of the oceanic plate. Reported and compiled data allow us to infer the continuity of the Carboniferous magmatic arc along the west margin of Gondwana.

Supplementary material: Detailed petrography, analytical methods and data, zircon cathodoluminescence images and supplementary figures are available at https://doi.org/10.6084/m9.figshare.c.4763993

Select agents (SA) pose unique challenges for licensing vaccines and therapies. In the case of toxin-mediated diseases, HHS assigns guidelines for SA use, oversees vaccine and therapy development, and approves animal models and approaches to identify mechanisms for toxin neutralization. In this commentary, we discuss next-generation vaccines and therapies against ricin toxin and botulinum toxin, which are regulated SA toxins that utilize structure-based approaches for countermeasures to guide rapid response to future biothreats.

Virus-like-particle (VLP) influenza vaccines can be given intramuscularly (i.m.) or intranasally (i.n.) and may have advantages over split-virion formulations in the elderly. We tested a plant-made VLP vaccine candidate bearing the viral hemagglutinin (HA) delivered either i.m. or i.n. in young and aged mice. Young adult (5- to 8-week-old) and aged (16- to 20-month-old) female BALB/c mice received a single 3-μg dose based on the HA (A/California/07/2009 H1N1) content of a plant-made H1-VLP (i.m. or i.n.) split-virion vaccine (i.m.) or were left naive. After vaccination, humoral and splenocyte responses were assessed, and some mice were challenged. Both VLP and split vaccines given i.m. protected 100% of the young animals, but the VLP group lost the least weight and had stronger humoral and cellular responses. Compared to split-vaccine recipients, aged animals vaccinated i.m. with VLP were more likely to survive challenge (80% versus 60%). The lung viral load postchallenge was lowest in the VLP i.m. groups. Mice vaccinated with VLP i.n. had little detectable immune response, but survival was significantly increased. In both age groups, i.m. administration of the H1-VLP vaccine elicited more balanced humoral and cellular responses and provided better protection from homologous challenge than the split-virion vaccine.

Reactive immunization with a single-dose cholera vaccine that could rapidly (within days) protect immunologically naive individuals during virgin soil epidemics, when cholera reaches immunologically naive populations that have not experienced cholera for decades, would facilitate cholera control. One dose of attenuated Vibrio cholerae O1 classical Inaba vaccine CVD 103-HgR (Vaxchora) containing ≥2 x 10⁸ CFU induces vibriocidal antibody seroconversion (a correlate of protection) in >90% of U.S. adults. A previous CVD 103-HgR commercial formulation required ≥2 x 10⁹ CFU to elicit high levels of seroconversion in populations in developing countries. We compared the vibriocidal responses of Malians (individuals 18 to 45 years old) randomized to ingest a single ≥2 x 10⁸-CFU standard dose (n = 50) or a ≥2 x 10⁹-CFU high dose (n = 50) of PaxVax CVD 103-HgR with buffer or two doses (n = 50) of Shanchol inactivated cholera vaccine (the immunologic comparator). To maintain blinding, participants were dosed twice 2 weeks apart; CVD 103-HgR recipients ingested placebo 2 weeks before or after ingesting vaccine. Seroconversion (a ≥4-fold vibriocidal titer rise) between the baseline and 14 days after CVD 103-HgR ingestion and following the first and second doses of Shanchol were the main outcomes measured. By day 14 postvaccination, the rates of seroconversion after ingestion of a single standard dose and a high dose of CVD 103-HgR were 71.7% (33/46 participants) and 83.3% (40/48 participants), respectively. The rate of seroconversion following the first dose of Shanchol, 56.0% (28/50 participants), was significantly lower than that following the high dose of CVD 103-HgR (P = 0.003). The vibriocidal geometric mean titer (GMT) of the high dose of CVD 103-HgR exceeded the GMT of the standard dose at day 14 (214 versus 95, P = 0.045) and was ~2-fold higher than the GMT on day 7 and day 14 following the first Shanchol dose (P > 0.05). High-dose CVD 103-HgR is recommended for accelerated evaluation in developing countries to assess its efficacy and practicality in field situations. (This study has been registered at ClinicalTrials.gov under registration no. NCT02145377.)

The kinetics, longevity, and breadth of antibodies to influenza virus neuraminidase (NA) in archival, sequential serum/plasma samples from influenza A virus (IAV) H5N1 infection survivors and from patients infected with the 2009 pandemic IAV (H1N1) virus were determined using an enzyme-linked lectin-based assay. The reverse-genetics-derived H4N1 viruses harboring a hemagglutinin (HA) segment from A/duck/Shan Tou/461/2000 (H4N9) and an NA segment derived from either IAV H5N1 clade 1, IAV H5N1 clade 2.3.4, the 2009 pandemic IAV (H1N1) (H1N1pdm), or A/Puerto Rico/8/1934 (H1N1) virus were used as the test antigens. These serum/plasma samples were also investigated by microneutralization (MN) and/or hemagglutination inhibition (HI) assays. Neuraminidase-inhibiting (NI) antibodies against N1 NA of both homologous and heterologous viruses were observed in H5N1 survivors and H1N1pdm patients. H5N1 survivors who were never exposed to H1N1pdm virus developed NI antibodies to H1N1pdm NA. Seroconversion of NI antibodies was observed in 65% of the H1N1pdm patients at day 7 after disease onset, but an increase in titer was not observed in serum samples obtained late in infection. On the other hand, an increase in seroconversion rate with the HI assay was observed in the follow-up series of sera obtained on days 7, 14, 28, and 90 after infection. The study also showed that NI antibodies are broadly reactive, while MN and HI antibodies are more strain specific.

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

We previously produced a heavy-chain-only antibody (Ab) VH domain (V_HH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific V_HHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the V_HH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique V_HHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 V_HHs grouped into more than 20 different competition bins. The RTA-specific V_HHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific V_HHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.

Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log₂; linear over a range of 4.27 to 9.65 log₂ 50% inhibitory concentration (IC₅₀); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, –30% to 30%), and total error (total error range, –65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

In December 2019, a novel coronavirus causing an infectious respiratory disease (COVID-19) was identified, which since then has developed into a pandemic with higher rates of mortality in older individuals and those with underlying medical conditions.¹ Multiple sclerosis (MS) is an immune-mediated neurologic disease which requires long-term treatment with immunotherapies that have been shown to increase the risk of infections.² As a result, there is significant anxiety among patients and neurologists during the pandemic regarding the infection outcome in this patient population. We present a patient with MS treated with fingolimod who was diagnosed with COVID-19 and had a favorable outcome.

In the article "Neurologic syndromes related to anti-GAD65: Clinical and serologic response to treatment" by Muñoz-Lopetegi et al.,¹ published online March 2, 2020, the y-axis label for figure 5’s right graph should be "CSF anti-GAD65 concentration (IU/mL)." The editorial office regrets the error.

Objective

To identify and characterize myeloid cell populations within the CSF of patients with MS and anti-myelin oligodendrocyte glycoprotein (MOG) disorder by high-resolution single-cell gene expression analysis.

Objective

To describe the presentations, radiologic features, and outcomes of children with autoimmune encephalitis associated with myelin oligodendrocyte glycoprotein antibodies (MOG abs).

Objective

To assess possible adverse effects on breastfed infants of mothers receiving monoclonal antibodies (MAbs) during pregnancy and/or lactation.

Objective

To determine the characteristic clinical and spinal MRI phenotypes of sarcoidosis-associated myelopathy (SAM), we analyzed a large cohort of patients with this disorder.

Alemtuzumab is approved for the treatment of relapsing-remitting MS and is used off-label for patients with chronic lymphocytic leukemia and as induction and antirejection therapy in kidney transplant recipients.¹ Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) complicating alemtuzumab treatment was reported in 9 patients with hematologic malignancy or MS.^1–3 The risk of GBS or CIDP in solid organ transplant recipients treated with alemtuzumab is unknown.

Objective

To provide first real-world experience on patients with MS treated with the B cell–depleting antibody ocrelizumab.

Transportation infrastructure owners manage an array of different asset types such as bridges, road pavements, earthworks and drainage. Currently, most organization management procedures are siloed by asset type; however, there are important interactions between these asset groups that need to be managed in a cross-asset way. Although these interactions are known, there is little or no quantification of these interactions. For the first time, this paper quantifies that 74% of Highways England's earthwork failures are a result of drainage-related problems, either the lack of drainage infrastructure or the poor performance of it. The analysis undertaken is an important first step not only in moving towards more connected asset management planning for earthworks and drainage, but to also provide guidance for other owners of earthwork infrastructure assets to improve their strategic asset management procedures.

Thematic collection: This article is part of the Ground-related risk to transportation infrastructure collection available at https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

In spite of the Kozeny–Carman formula having been applied effectively to sands, there is at present no generally accepted explanation for why it is inadequate for clays. The impermeable adsorbed water layer surrounding the soil particles throws further light on the issue. By introducing the pervious void ratio, which accounts for the effective voids that a fluid flows through, a modified Kozeny–Carman formula is presented. The results presented here show that the modified Kozeny–Carman formula predicts fairly well the hydraulic conductivity of clays, especially for fine-grained soils.

The presence of large palaeo-landslides can hinder the construction of railway lines if they cause an alteration of the natural balance of the slope, significantly increasing the cost of the project. During the construction works in a section of the Madrid–Asturias high-speed railway line (Spain), a large-scale hillside instability affected 460 m of the section. The ground movement began 10 months after the start of the excavations and remained active throughout the observation period (2008–2010). Data provided by fieldwork, boreholes and instrumental monitoring have allowed the investigation of the geological units involved, together with the geometry and the kinematics of the mass movement. This landslide involves a Paleozoic basement with an estimated volume of 4 400 000 m³. It shows low displacement rates (<45 mm a^–1) and was accommodated on a single surface of rupture, which reaches more than 60 m depth. The kinematics is favoured by structural and lithological conditions, there being a strong rheological contrast in the lower part of the mobilized ground. This movement is linked to a large palaeo-landslide that was partially reactivated when excavations undermined the lower slopes of the hillside. Technical solutions for the stabilization have already caused an additional cost of 17% over the initial budget.

Loess, a wind-blown silty soil, can be deposited under a variety of moisture conditions, including dry deposition, wet deposition and gravitational settling of aggregations formed in moist air by capillary forces at grain contacts. This experimental study uses single and double oedometer tests to assess the effect of depositional water content on the collapse potential of reconstituted samples of the Langley Silt Member, known as Brickearth, a natural loessic soil. A freefall sample preparation technique was used to mimic loess formation and environmental scanning electron microscopy was used to relate the observed behaviour to sample fabric. The results show that loess deposited at higher water contents has a greater collapse potential, which is shown to be related to its looser, more granular fabric.

This case study aims to identify the cubic capacity and geometry of the geological body of silt–organic sediments in the environment of a former gravel pit situated in a drainless depression of the alluvium of the Čierna voda River. It is located in the well-known geotourism locality of Slnečné Jazera in Senec, in the SW of Slovakia. To identify the body, electrical resistivity tomography was combined with the use of sonar. The research shows that this approach is appropriate for a number of activities that are subjects of engineering-geological investigations. The cubic capacity and geometry of specific aqueous engineering-geological environments must be determined in connection with the need for the management, control, quantification and extraction of selected sedimentary bodies. In addition, the management of sustainable development of reservoirs, sedimentation basins, industrial ponds, settling pits and natural pools for recreation (the subject of the case study) is important to control the limit amounts of sediments in such environments. The results of this study may be applied in analogous engineering-geological conditions. The drainless depression Slnečné Jazera contained a body of silt–organic sediments amounting to 23 000 m³ (41 Olympic-size pools of 25 m x 12.5 m x 1.8 m). The maximum thickness of the bottom sediments was about 6.3 m on the alluvium with an articulated morphology owing to the submerged digging of gravel. The proposed approach improved the control of extraction of the sedimentary body and optimized the engineering-geological conditions in this geotourism locality.

This paper uses a 3D model for stability assessment of Zhujiabaobao waste dump with ground cracks. The study data were gathered via reconnaissance, geomorphological analysis and laboratory experiment. A 3D finite extended element method model that can consider cracks was then used to calculate the factor of safety (FOS) of the waste dump via the strength reduction technique. The simulation shows the dump to have an FOS of 1.22 and both the position and depth of penetration of cracks in the waste dump have a crucial impact on the stability of the slope. Because the study area is located in a seismically active area, simulation and analysis of the dynamic response of the waste dump under different magnitudes of seismic waves (peak acceleration is 0.05, 0.15, 0.25 and 0.45g) were performed via an explicit dynamic model. The simulation shows that high steps in the slope are particularly responsive to earthquakes. The approach used here for analysing stability under static and dynamic loads is useful for hazard prevention and mitigation.

The decline or drying up of groundwater sources near a tunnel route is damaging to groundwater users. Therefore, forecasting the impact of a tunnel on nearby groundwater sources is a challenging task in tunnel design. In this study, numerical and analytical approaches were applied to the Qomroud water conveyance tunnel (located in Lorestan province, Iran) to assess the impact of tunnelling on the nearby extraction water wells. Using simulation of groundwater-level fluctuation owing to tunnelling, the drawdown at the well locations was determined. From the drawdowns and using Dupuit's equation, the depletion of well flow rates after tunnelling was estimated. To evaluate the results, observed well flow rates before and after tunnelling were compared with the predicted flow rates. The observed and estimated water well flows (before and after tunnelling) showed a regression factor of 0.64, pointing to satisfactory results

The Clay-with-flints Formation outcrops on the high chalk plateaux and interfluves of the chalk downs in southern England. Both current and historical definitions of the Clay-with-flints are detailed and important distinctions are identified with other deposits that appear identical but are formed in different ways. Historically pedological or geomorphological studies have been carried out on the deposit. Engineering studies are only carried out where the deposit is crossed by infrastructure. The physical and chemical processes acting on the deposit and the resulting effects on the physical properties are discussed.

In this paper, a decision tree is presented, constructed on the basis of hydrogeological characteristics (water table depth, freshwater thickness, surface area required and distance between wells), to choose the optimal groundwater extraction method in the case of a coastal unconfined aquifer. A comparison is made of the groundwater extraction methods in a freshwater aquifer of limited thickness occurring in coastal dunes in the eastern region of the Province of Buenos Aires (Argentina). The negative effects brought about by the wrong use of the groundwater extraction methods are analysed, because, as a result of excessive extraction, such methods lead to the dramatic decrease of the freshwater reserves. The decision tree is a useful tool to assist decision-makers as it suggests the most suitable groundwater extraction method options (vertical wells or wellpoints), as well as identifying areas that are unsuitable for sustainable groundwater extraction.

A logistic regression model and a bivariate statistical analysis were used in this paper to evaluate the groundwater recharge susceptibility. The approach is based on the assessment of the relationship involving groundwater recharge and parameters that influence this hydrological process. Surface parameters and aquifer-related parameters were evaluated as thematic map layers using ArcGIS. Then, a weighted-rating method was adopted to categorize each parameter's map. To assess the role of each parameter in the aquifer recharge, a logistic regression model and a bivariate statistical analysis were applied to the Guenniche phreatic aquifer (Tunisia). Models are explored to establish a map showing the aquifer recharge susceptibility. The code Modflow was used to simulate the consequence of the recharge. The recharge amount was introduced in the model and was tested to verify the recharge effect on the hydraulic head for the two models. The obtained results reveal that the recharge as mapped in the bivariate statistical model has a minor impact on the hydraulic head. Results of the logistic regression model are more significant as the hydraulic head is widely affected. This model provides good results in mapping the spatial distribution of the aquifer recharge susceptibility.

In 2015, the M_w 7.8 Gorkha earthquake struck Nepal, triggering thousands of landslides across the central and eastern Himalayas. These landslides had many adverse effects, including causing widespread damage to low-grade transport routes (e.g. tracks, footpaths) in rural regions that depend on tourism for survival. Langtang Valley is a glacial–periglacial landscape located 60 km north of Kathmandu. It is one of the most popular trekking regions in Nepal and has been severely affected by Gorkha earthquake-triggered and monsoon-triggered landsliding. Here, qualitative and quantitative observations from fieldwork and remote sensing are used to describe the materials and geomorphology of the landslides across Langtang Valley, and to quantify the extent to which coseismic and monsoon-triggered landslides have affected Langtang's trekking infrastructure. The dominant bedrock materials involved within Langtang landslides are found to be a range of gneisses and intruded leucogranites. In total, 64 landslides are found to have intersected trekking paths across Langtang, with coseismic and monsoon-triggered landslides having an impact on c. 3 km and 0.8 km of path respectively. It is observed that the practice of reconstructing paths through unstable landslide deposits is leaving the trekking infrastructure across Langtang increasingly vulnerable to future failure.

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

Screening for Chlamydia trachomatis and Neisseria gonorrhoeae at the pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Combining the three individual-site samples into a single pooled sample could result in significant cost savings, provided there is no significant sensitivity reduction. The aim of this study was to examine the sensitivity of pooled samples for detecting chlamydia and gonorrhea in asymptomatic MSM using a nucleic acid amplification test. Asymptomatic MSM who tested positive for chlamydia or gonorrhoea were invited to participate. Paired samples were obtained from participants prior to administration of treatment. To form the pooled sample, the anorectal swab was agitated in the urine specimen transport tube and then discarded. The pharyngeal swab and 2 ml of urine sample were then added to the tube. The difference in sensitivity between testing of pooled samples and individual-site testing was calculated against an expanded gold standard, where an individual is considered positive if either pooled-sample or individual-site testing returns a positive result. All samples were tested using the Aptima Combo 2 assay. A total of 162 MSM were enrolled in the study. Sensitivities of pooled-sample testing were 86% (94/109; 95% confidence interval [CI], 79 to 92%]) for chlamydia and 91% (73/80; 95% CI, 83 to 96%) for gonorrhea. The sensitivity reduction was significant for chlamydia (P = 0.02) but not for gonorrhea (P = 0.34). Pooling caused 22 infections (15 chlamydia and 7 gonorrhoea) to be missed, and the majority were single-site infections (19/22). Pooling urogenital and extragenital samples from asymptomatic MSM reduced the sensitivity of detection by approximately 10% for chlamydia but not for gonorrhea.

Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (C_T), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median C_T was lower in toxin-positive samples than in toxin-negative samples; however, C_T ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

The number of onsite clinical microbiology laboratories in hospitals is decreasing, likely related to the business model for laboratory consolidation and labor shortages, and this impacts a variety of clinical practices, including that of banking isolates for clinical or epidemiologic purposes. To determine the impact of these trends, infectious disease (ID) physicians were surveyed regarding their perceptions of offsite services. Clinical microbiology practices for retention of clinical isolates for future use were also determined. Surveys were sent to members of the Infectious Diseases Society of America’s (IDSA) Emerging Infections Network (EIN). The EIN is a sentinel network of ID physicians who care for adult and/or pediatric patients in North America and who are members of IDSA. The response rate was 763 (45%) of 1,680 potential respondents. Five hundred forty (81%) respondents reported interacting with the clinical microbiology laboratory. Eighty-six percent of respondents thought an onsite laboratory very important for timely diagnostic reporting and ongoing communication with the clinical microbiologist. Thirty-five percent practiced in institutions where the core microbiology laboratory has been moved offsite, and an additional 7% (n = 38) reported that movement of core laboratory functions offsite was being considered. The respondents reported that only 24% of laboratories banked all isolates, with the majority saving isolates for less than 30 days. Based on these results, the trend toward centralized core laboratories negatively impacts the practice of ID physicians, potentially delays effective implementation of prompt and targeted care for patients with serious infections, and similarly adversely impacts infection control epidemiologic investigations.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

There are roughly 48,000 deaths caused by influenza annually and an estimated 200,000 people who have undiagnosed human immunodeficiency virus (HIV). These are examples of acute and chronic illnesses that can be identified by employing a CLIA-waived test. Pharmacies across the country have been incorporating CLIA-waived point-of-care tests (POCT) into disease screening and management programs offered in the pharmacy. The rationale behind these programs is discussed. Additionally, a summary of clinical data for some of these programs in the infectious disease arena is provided. Finally, we discuss the future potential for CLIA-waived POCT-based programs in community pharmacies.

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy.

On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID₅₀]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 x 10⁴ RNA copies/ml (range, 2.21 x 10² to 4.71 x 10⁵ RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman’s correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

A time course experiment is a widely used design in the study of cellular processes such as differentiation or response to stimuli. In this paper, we propose time course regulatory analysis (TimeReg) as a method for the analysis of gene regulatory networks based on paired gene expression and chromatin accessibility data from a time course. TimeReg can be used to prioritize regulatory elements, to extract core regulatory modules at each time point, to identify key regulators driving changes of the cellular state, and to causally connect the modules across different time points. We applied the method to analyze paired chromatin accessibility and gene expression data from a retinoic acid (RA)–induced mouse embryonic stem cells (mESCs) differentiation experiment. The analysis identified 57,048 novel regulatory elements regulating cerebellar development, synapse assembly, and hindbrain morphogenesis, which substantially extended our knowledge of cis-regulatory elements during differentiation. Using single-cell RNA-seq data, we showed that the core regulatory modules can reflect the properties of different subpopulations of cells. Finally, the driver regulators are shown to be important in clarifying the relations between modules across adjacent time points. As a second example, our method on Ascl1-induced direct reprogramming from fibroblast to neuron time course data identified Id1/2 as driver regulators of early stage of reprogramming.

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100x higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.