SARS-CoV-2 Delta and Omicron community transmission networks as added value to contact tracing.

OBJECTIVES: Calculations of SARS-CoV-2 transmission networks at a population level have been limited. Networks that estimate infections between individuals and whether this results in a mutation, can be a way to evaluate fitness of a mutational clone by how much it expands in number as well as determining the likelihood a transmission results in a new variant. METHODS: Australian Delta and Omicron SARS-CoV-2 sequences were downloaded from GISAID. Transmission networks of infection between individuals were estimated using a novel mathematical method. RESULTS: Many of the sequences were identical, with clone sizes following power law distributions driven by negative binomial probability distributions for both the number of infections per individual and the number of mutations per transmission (median 0.74 nucleotide changes for Delta and 0.71 for Omicron). Using these distributions, an agent-based model was able to replicate the observed clonal network structure, providing a basis for more detailed COVID-19 modelling. Possible recombination events, tracked by insertion/deletion (indel) patterns, were identified for each variant in these outbreaks. CONCLUSIONS: This modelling approach reveals key transmission characteristics of SARS-CoV-2 and may complement traditional contact tracing. This methodology can also be applied to other diseases as genetic sequencing of viruses becomes more commonplace.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasite Toxoplasma gondii.

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ∼five-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.

Benefits and harms of cervical screening, triage and treatment strategies in women living with HIV.

To support a strategy to eliminate cervical cancer as a public health problem, the World Health Organisation (WHO) reviewed its guidelines for screening and treatment of cervical pre-cancerous lesions in 2021. Women living with HIV have 6-times the risk of cervical cancer compared to women in the general population, and we harnessed a model platform ('Policy1-Cervix-HIV') to evaluate the benefits and harms of a range of screening strategies for women living with HIV in Tanzania, a country with endemic HIV. Assuming 70% coverage, we found that 3-yearly primary HPV screening without triage would reduce age-standardised cervical cancer mortality rates by 72%, with a number needed to treat (NNT) of 38.7, to prevent a cervical cancer death. Triaging HPV positive women before treatment resulted in minimal loss of effectiveness and had more favorable NNTs (19.7-33.0). Screening using visual inspection with acetic acid (VIA) or cytology was less effective than primary HPV and, in the case of VIA, generated a far higher NNT of 107.5. These findings support the WHO 2021 recommendation that women living with HIV are screened with primary HPV testing in a screen-triage-and-treat approach starting at 25 years, with regular screening every 3-5 years.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasite Toxoplasma gondii.

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ∼ 5-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.

ENTRAIN: integrating trajectory inference and gene regulatory networks with spatial data to co-localize the receptor-ligand interactions that specify cell fate.

MOTIVATION: Cell fate is commonly studied by profiling the gene expression of single cells to infer developmental trajectories based on expression similarity, RNA velocity, or statistical mechanical properties. However, current approaches do not recover microenvironmental signals from the cellular niche that drive a differentiation trajectory. RESULTS: We resolve this with environment-aware trajectory inference (ENTRAIN), a computational method that integrates trajectory inference methods with ligand-receptor pair gene regulatory networks to identify extracellular signals and evaluate their relative contribution towards a differentiation trajectory. The output from ENTRAIN can be superimposed on spatial data to co-localize cells and molecules in space and time to map cell fate potentials to cell-cell interactions. We validate and benchmark our approach on single-cell bone marrow and spatially resolved embryonic neurogenesis datasets to identify known and novel environmental drivers of cellular differentiation. AVAILABILITY AND IMPLEMENTATION: ENTRAIN is available as a public package at https://github.com/theimagelab/entrain and can be used on both single-cell and spatially resolved datasets.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here, we address the role of the cortical microtubules in parasite motility, invasion and egress by utilizing a previously generated mutant (dubbed 'TKO') in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ∼80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization was further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication was not affected. In a 3D Matrigel matrix, the TKO mutant moved directionally over long distances, but along trajectories that were significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory did not impact either movement speed in the matrix or the speed and behavior of the parasite during entry into and egress from the host cell.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3-D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here we address the role of the cortical microtubules in parasite motility, invasion, and egress by utilizing a previously generated mutant (dubbed "TKO") in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ~ 80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization is further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication is not affected. In a 3-D Matrigel matrix, the TKO mutant moves directionally over long distances, but along trajectories significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory does not impact either movement speed in the matrix or the speed and behavior of the parasite's entry into and egress from the host cell.

SARS-CoV-2 Variability in Patients and Wastewaters-Potential Immuno-Modulation during the Shift from Delta to Omicron.

The continuous emergence of SARS-CoV-2 variants favors potential co-infections and/or viral mutation events, leading to possible new biological properties. The aim of this work was to characterize SARS-CoV-2 genetic variability during the Delta-Omicron shift in patients and in a neighboring wastewater treatment plant (WWTP) in the same urban area. The surveillance of SARS-CoV-2 was performed by routine screening of positive samples by single nucleotide polymorphism analysis within the S gene. Moreover, additionally to national systematic whole genome sequencing (WGS) once a week in SARS-CoV-2-positive patients, WGS was also applied when mutational profiles were difficult to interpret by routine screening. Thus, WGS was performed on 414 respiratory samples and on four wastewater samples, northeastern France. This allowed us to report (i) the temporally concordant Delta to Omicron viral shift in patients and wastewaters; (ii) the characterization of 21J (Delta) and 21K (Omicron)/BA.1-21L (Omicron)/BA.2-BA.4 mixtures from humans or environmental samples; (iii) the mapping of composite mutations and the predicted impact on immune properties in the viral Spike protein.

An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

Dynamics of latent HIV under clonal expansion.

The HIV latent reservoir exhibits slow decay on antiretroviral therapy (ART), impacted by homeostatic proliferation and activation. How these processes contribute to the total dynamic while also producing the observed profile of sampled latent clone sizes is unclear. An agent-based model was developed that tracks individual latent clones, incorporating homeostatic proliferation of cells and activation of clones. The model was calibrated to produce observed latent reservoir dynamics as well as observed clonal size profiles. Simulations were compared to previously published latent HIV integration data from 5 adults and 3 children. The model simulations reproduced reservoir dynamics as well as generating residual plasma viremia levels (pVL) consistent with observations on ART. Over 382 Latin Hypercube Sample simulations, the median latent reservoir grew by only 0.3 log10 over the 10 years prior to ART initiation, after which time it decreased with a half-life of 15 years, despite number of clones decreasing at a faster rate. Activation produced a maximum size of genetically intact clones of around one million cells. The individual simulation that best reproduced the sampled clone profile, produced a reservoir that decayed with a 13.9 year half-life and where pVL, produced mainly from proliferation, decayed with a half-life of 10.8 years. These slow decay rates were achieved with mean cell life-spans of only 14.2 months, due to expansion of the reservoir through proliferation and activation. Although the reservoir decayed on ART, a number of clones increased in size more than 4,000-fold. While small sampled clones may have expanded through proliferation, the large sizes exclusively arose from activation. Simulations where homeostatic proliferation contributed more to pVL than activation, produced pVL that was less variable over time and exhibited fewer viral blips. While homeostatic proliferation adds to the latent reservoir, activation can both add and remove latent cells. Latent activation can produce large clones, where these may have been seeded much earlier than when first sampled. Elimination of the reservoir is complicated by expanding clones whose dynamic differ considerably to that of the entire reservoir.

High Rates of Liver Cirrhosis and Hepatocellular Carcinoma in Chronic Hepatitis B Patients with Metabolic and Cardiovascular Comorbidities.

BACKGROUND: The prevalence of metabolic and cardiovascular diseases is rising worldwide. However, little is known about the impact of such disorders on hepatic disease progression in chronic hepatitis B (CHB) during the era of potent nucleo(s)tide analogues (NAs). METHODS: We retrospectively analyzed a single-center cohort of 602 CHB patients, comparing the frequency of liver cirrhosis at baseline and incidences of liver-related events during follow-up (hepatocellular carcinoma, liver transplantation and liver-related death) between CHB patients with a history of diabetes, obesity, hypertension or coronary heart disease (CHD). RESULTS: Rates of cirrhosis at baseline and liver-related events during follow-up (median follow-up time: 2.51 years; NA-treated: 37%) were substantially higher in CHB patients with diabetes (11/23; 3/23), obesity (6/13; 2/13), CHD (7/11; 2/11) or hypertension (15/43; 4/43) compared to CHB patients without the indicated comorbidities (26/509; 6/509). Multivariate analysis identified diabetes as the most significant predictor for cirrhosis (p = 0.0105), while comorbidities did not correlate with liver-related events in pre-existing cirrhosis. CONCLUSION: The combination of metabolic diseases and CHB is associated with substantially increased rates of liver cirrhosis and secondary liver-related events compared to CHB alone, indicating that hepatitis B patients with metabolic comorbidities warrant particular attention in disease surveillance and evaluation of treatment indication.

Birth-cohort estimates of smoking initiation and prevalence in 20th century Australia: Synthesis of data from 33 surveys and 385,810 participants.

The aim of our study was to quantify sex-specific patterns of smoking prevalence and initiation in 10-year birth cohorts from 1910 to 1989 in Australia. We combined individual data of 385,810 participants from 33 cross-sectional surveys conducted between 1962 and 2018. We found that age-specific smoking prevalence varied considerably between men and women within birth cohorts born before 1960. The largest difference was observed in the earliest cohort (1910-1919), with up to 37.7% point greater proportion of current smokers in men than in women. In subsequent cohorts, the proportion decreased among men, but increased among women, until there was no more than 7.4% point difference in the 1960-69 birth cohort. In the 1970-79 and 1980-89 cohorts, smoking among men marginally increased, but the proportion was at most ~11.0% points higher than women. Our analysis of initiation indicated that many women born before the 1930s who smoked commenced smoking after age 25 years (e.g., ~27% born in 1910-19); compared to at most 8% of men in any birth cohort. The earliest birth cohort (1910-1919) had the greatest difference in age at initiation between sexes; 26.6 years in women versus 19.0 in men. In later cohorts, male and female smokers initiated increasingly earlier, converging in the 1960-69 cohort (17.6 and 17.8 years, respectively). While 22.9% of men and 8.4% of women initiated smoking aged < = 15 in the 1910-1919 cohort, in the latest cohort (1980-89) the reverse was true (21.4% and 28.8% for men and women, respectively). Marked differences in smoking prevalence and age at initiation existed between birth cohorts of Australian men and women born before 1960; after this, sex-specific trends in prevalence and initiation were similar. Understanding these patterns may inform the evaluation of tobacco control policies and the targeting of potential interventions for exposed populations such as lung cancer screening.

Variability in molecular characteristics of Hepatitis E virus quasispecies could modify viral surface properties and transmission.

Hepatitis E virus (HEV) usually causes self-limited liver diseases but can also result in severe cases. Genotypes 1 (G1) and 2 circulate in developing countries are human-restricted and waterborne, while zoonotic G3 and G4 circulating in industrialized countries preferentially infect human through consumption of contaminated meat. Our aims were to identify amino acid patterns in HEV variants that could be involved in pathogenicity or in transmission modes, related to their impact on antigenicity and viral surface hydrophobicity. HEV sequences from human (n = 37) and environmental origins (wild boar [n = 3], pig slaughterhouse effluent [n = 6] and urban wastewater [n = 2]) were collected for the characterization of quasispecies using ultra-deep sequencing (ORF2/ORF3 overlap). Predictive and functional assays were carried out to investigate viral particle antigenicity and hydrophobicity. Most quasispecies showed a major variant while a mixture was observed in urban wastewater and in one chronically infected patient. Amino acid signatures were identified, as a rabbit-linked HEV pattern in two infected patients, or the S68L (ORF2) / H81C (ORF3) residue mostly identified in wild boars. By comparison with environmental strains, molecular patterns less likely represented in humans were identified. Patterns impacting viral hydrophobicity and/or antigenicity were also observed, and the higher hydrophobicity of HEV naked particles compared with the enveloped forms was demonstrated. HEV variants isolated from human and environment present molecular patterns that could impact their surface properties as well as their transmission. These molecular patterns may concern only one minor variant of a quasispecies and could emerge under selective pressure.

NGlyAlign: an automated library building tool to align highly divergent HIV envelope sequences.

BACKGROUND: The high variability in envelope regions of some viruses such as HIV allow the virus to establish infection and to escape subsequent immune surveillance. This variability, as well as increasing incorporation of N-linked glycosylation sites, is fundamental to this evasion. It also creates difficulties for multiple sequence alignment methods (MSA) that provide the first step in their analysis. Existing MSA tools often fail to properly align highly variable HIV envelope sequences requiring extensive manual editing that is impractical with even a moderate number of these variable sequences. RESULTS: We developed an automated library building tool NGlyAlign, that organizes similar N-linked glycosylation sites as block constraints and statistically conserved global sites as single site constraints to automatically enforce partial columns in consistency-based MSA methods such as Dialign. This combined method accurately aligns variable HIV-1 envelope sequences. We tested the method on two datasets: a set of 156 founder and chronic gp160 HIV-1 subtype B sequences as well as a set of reference sequences of gp120 in the highly variable region 1. On measures such as entropy scores, sum of pair scores, column score, and similarity heat maps, NGlyAlign+Dialign proved superior against methods such as T-Coffee, ClustalOmega, ClustalW, Praline, HIValign and Muscle. The method is scalable to large sequence sets producing accurate alignments without requiring manual editing. As well as this application to HIV, our method can be used for other highly variable glycoproteins such as hepatitis C virus envelope. CONCLUSIONS: NGlyAlign is an automated tool for mapping and building glycosylation motif libraries to accurately align highly variable regions in HIV sequences. It can provide the basis for many studies reliant on single robust alignments. NGlyAlign has been developed as an open-source tool and is freely available at https://github.com/UNSW-Mathematical-Biology/NGlyAlign_v1.0 .

Elimination of cervical cancer in Tanzania: Modelled analysis of elimination in the context of endemic HIV infection and active HIV control.

The World Health Organisation (WHO) has launched a strategic initiative for cervical cancer (CC) elimination which involves scaling up three interventions: human papillomavirus (HPV) vaccination, twice-lifetime HPV-screening screening and pre-cancer/cancer treatment by 2030. CC is challenging to control in countries with endemic human immunodeficiency virus (HIV), as women living with HIV (WLHIV) are at elevated risk of HPV infection, persistence and progression. This analysis estimated the impact of the elimination interventions on CC incidence and mortality but additionally considered more intensive screening for WLHIV, using Tanzania as an example. A dynamic HIV/HPV model was used to simulate the elimination strategy for vaccination, screening and pre-cancer/cancer treatment, with 3-yearly HPV-screening in WLHIV starting at age 25 years, in the context of sustained HIV control in Tanzania from 2020 to 2119. Without vaccination or HPV screening, CC incidence rates per 100 000 women are predicted to fall from 58.0 in 2020 to 41.6 (range: 39.1-44.7) in 2119, due to existing HIV control. HPV vaccination and twice-lifetime HPV-screening for the general population and 3-yearly screening for WLHIV, would reduce CC incidence to 1.3 (range: 1.3-2.5) by 2119, with elimination (<4/100 000) in 2076 (range: 2076-2092). CC mortality rates per 100 000 women are predicted to reach 1.1 (range: 1.1-2.1) with further reductions contingent on increased CC treatment access. Vaccination and 3-yearly HPV-screening for WLHIV is predicted to achieve elimination in the subgroup of WLHIV potentially as early as 2061 (range: 2061-2078), with a 2119 CC incidence rate of 1.7 (range: 1.7-3.3). Scaling-up vaccination and HPV-screening will substantially reduce CC incidence in Tanzania, with elimination predicted within a century. Three-yearly HPV-screening and HPV vaccination, at high coverage rates, would facilitate CC elimination among WLHIV, and thus accelerate elimination in the overall population.

Mutational networks of escape from transmitted HIV-1 infection.

Human immunodeficiency virus (HIV) is subject to immune selective pressure soon after it establishes infection at the founder stage. As an individual progresses from the founder to chronic stage of infection, immune pressure forces a history of mutations that are embedded in envelope sequences. Determining this pathway of coevolving mutations can assist in understanding what is different with the founder virus and the essential pathways it takes to maintain infection. We have combined operations research and bioinformatics methods to extract key networks of mutations that differentiate founder and chronic stages for 156 subtype B and 107 subtype C envelope (gp160) sequences. The chronic networks for both subtypes revealed strikingly different hub-and-spoke topologies compared to the less structured transmission networks. This suggests that the hub nodes are impacted by the immune response and the resulting loss of fitness is compensated by mutations at the spoke positions. The major hubs in the chronic C network occur at positions 12, 137 (within the N136 glycan), and 822, and at position 306 for subtype B. While both founder networks had a more heterogeneous connected network structure, interestingly founder B subnetworks around positions 640 and 837 preferentially contained CD4 and coreceptor binding domains. Finally, we observed a differential effect of glycosylation between founder and chronic subtype B where the latter had mutational pathways significantly driven by N-glycosylation. Our study provides insights into the mutational pathways HIV takes to evade the immune response, and presents features more likely to establish founder infection, valuable for effective vaccine design.

The past, present and future impact of HIV prevention and control on HPV and cervical disease in Tanzania: A modelling study.

BACKGROUND: Women with HIV have an elevated risk of HPV infection, and eventually, cervical cancer. Tanzania has a high burden of both HIV and cervical cancer, with an HIV prevalence of 5.5% in women in 2018, and a cervical cancer incidence rate among the highest globally, at 59.1 per 100,000 per year, and an estimated 9,772 cervical cancers diagnosed in 2018. We aimed to quantify the impact that interventions intended to control HIV have had and will have on cervical cancer in Tanzania over a period from 1995 to 2070. METHODS: A deterministic transmission-dynamic compartment model of HIV and HPV infection and natural history was used to simulate the impact of voluntary medical male circumcision (VMMC), anti-retroviral therapy (ART), and targeted pre-exposure prophylaxis (PrEP) on cervical cancer incidence and mortality from 1995-2070. FINDINGS: We estimate that VMMC has prevented 2,843 cervical cancer cases and 1,039 cervical cancer deaths from 1995-2020; by 2070 we predict that VMMC will have lowered cervical cancer incidence and mortality rates by 28% (55.11 cases per 100,000 women in 2070 without VMMC, compared to 39.93 with VMMC only) and 26% (37.31 deaths per 100,000 women in 2070 without VMMC compared to 27.72 with VMMC), respectively. We predict that ART will temporarily increase cervical cancer diagnoses and deaths, due to the removal of HIV death as a competing risk, but will ultimately further lower cervical cancer incidence and mortality rates by 7% (to 37.31 cases per 100,000 women in 2070) and 5% (to 26.44 deaths per 100,000 women in 2070), respectively, relative to a scenario with VMMC but no ART. A combination of ART and targeted PrEP use is anticipated to lower cervical cancer incidence and mortality rates to 35.82 and 25.35 cases and deaths, respectively, per 100,000 women in 2070. CONCLUSIONS: HIV treatment and control measures in Tanzania will result in long-term reductions in cervical cancer incidence and mortality. Although, in the near term, the life-extending capability of ART will result in a temporary increase in cervical cancer rates, continued efforts towards HIV prevention will reduce cervical cancer incidence and mortality over the longer term. These findings are critical background to understanding the longer-term impact of achieving cervical cancer elimination targets in Tanzania.

A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers.

BACKGROUND: TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. RESULTS: We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. CONCLUSIONS: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.

BACKGROUND: Chronic hepatitis delta virus (HDV) infection causes severe liver disease which often leads to cirrhosis and hepatocellular carcinoma (HCC). Aim of this study was to establish the disease severity and prognostic factors for disease outcome by analysing frequencies of clinical events and their correlation with baseline virological and biochemical parameters as well as interferon and nucleos(t)ide analogue treatment choice. METHODS: We studied a single-centre cohort of 49 anti-HDAg-positive patients with HBsAg persistence for at least 6 months. Virological and biochemical parameters, interferon and nucleos(t)ide analogue treatment choice as well as clinical events during follow-up were analysed by retrospective chart review (mean follow-up time 3 years, range 0.25-7.67 years). RESULTS: Severe clinical events occurred in 11/49 hepatitis D patients, including HCC (8/49), death (8/49) or liver transplantation (2/49). HCCs only occurred secondary to liver cirrhosis and their event rates in this cohort of hepatitis D patients did not differ from a matched HBV mono-infected cohort with comparable frequency of liver cirrhosis. A stepwise multivariate logistic regression revealed low platelet count (p = 0. 0290) and older age (p = 0.0337) correlating most strongly with overall clinical events, while serum HDV RNA positivity at baseline did not correlate with any clinical outcome. Interferon-free but not nucleos(t)ide analogue-free patient care correlated with the occurrence of HCC at logistic regression, although only 3/18 interferon-treated patients demonstrated repeatedly negative HDV PCR results post therapy. CONCLUSIONS: Our data indicate that progressive liver disease at baseline plays a major role as predictive factor for overall clinical outcome of hepatitis D patients. In particular, HCC risk may not be underestimated in hepatitis D virus RNA negative hepatitis D patients with advanced liver fibrosis.

Treatment as prevention enrolling at least 75% of individuals on ART will be needed to significantly reduce HIV prevalence in a HIV cohort.

BACKGROUND: "Treatment as Prevention" (TasP) aims to reduce new HIV infections through higher enrolment on suppressive antiretroviral therapy (ART). OBJECTIVES: We studied the current epidemic and possible impact of TasP in a French HIV cohort including MSM and migrant subjects. STUDY DESIGN: Socio-demographic, clinical and laboratory variables were collected during the follow-up of 6995 HIV-infected patients. The numbers of individuals living with HIV in each year were estimated from diagnoses up to that year minus recorded deaths. Patients were classified according to gender, transmission mode, country of birth and treatment status. RESULTS: The cohort includes 6995 individuals diagnosed from 1985 to 2015, of whom 72% were men. Unprotected sexual intercourse was the main mode of transmission. Women were more likely to be migrants (45% versus 13%), whereas men were more likely to have been born in France (52% versus 27%). Diagnoses were more correlated with untreated than treated prevalence in each group. MSM diagnoses was strongly correlated to untreated prevalence whatever the country of birth (p < 0.0001). However, heterosexual diagnoses were better correlated with prevalence within individual country groups (b = 0.29 female diagnoses/year per untreated male born in France, compared to b = 0.73 for foreigners). Using these transmission rates, mathematical modelling estimated that enrolling 75% of untreated individuals per year would decrease diagnoses ten-fold by 2021. CONCLUSIONS: Enrolling at least 75% of individuals on ART is necessary to substantially impact numbers of new HIV infections in this cohort. Treatment as prevention will actually be effective to reduce HIV prevalence.