Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite  Plasmodium, Giardia and the kinetoplastids, including Leishmania spp., Trypanosoma cruzi, and Trypanosoma congolense  images, the haptomonad form attachment plaque is strikingly reminiscent of the hemidesmosomes that attach epithelial cells to the underlying extracellular matrix. Hemidesmosomes are formed by a set of transmembrane proteins (integrins and BP180), which connect the extracellular basal lamina to intracellular adaptor proteins (Plectin and BP230) that bind to intermediate filaments . The assL. mexicana infected sand flies and in-vitro-derived haptomonad-like cells attached to surfaces, with haptomonads being defined by their morphology and the formation of an attachment plaque  approaches to image haptomonad forms in the sand fly, enabling us to synthesise a unified view of L. mexicana haptomonads colonising the stomodeal valve, we used serial block face scanning electron microscopy (SBFSEM) to examine fixed and dissected midguts from sand flies that had been infected with L. mexicana for 10 days  to the stomodeal valve surface and connecting structures spanning this gap between the flagellar membrane and the putative wax layer of the stomodeal valve were seen . Within The haptomonad flagellar membrane did not encase the flagellum smoothly, and there were numerous projections and indentations within the membrane . FurtherIn vitro haptomonad-like cells have previously been generated by allowing promastigote cells to adhere to scratched plastic . We usedWe used serial section electron tomography to examine the 3D ultrastructure of the in vitro haptomonad-like cells. The overall organisation of the attachment structure of the in vitro haptomonad-like cell was highly similar to that of the sand fly haptomonads. The in vitro cells had a shortened attached flagellum with a 9+2 microtubule axoneme, without the PFR, a shorter and a wider anterior cell tip, with an expanded FAZ area containing typical junctional complexes . FilamenIn addition, we compared the morphology of in vitro haptomonad-like cells with the sand fly haptomonads by measuring flagellum and axoneme length, and cell body length and width using the SBFSEM data . The in The plastic surface used for generation of in vitro haptomonad-like cell was suitable for electron microscopy analyses, but was not suitable for light microscopy. However, we discovered that specific glass substrates whose surfaces have hydrophobic properties supported the differentiation of promastigotes to in vitro haptomonad-like cells. We took advantage of this and followed the in vitro adhesion process using time-lapse microscopy . The adhWhen examining cells that had been allowed to adhere to glass for 24 hr, we identified each of these stages described above by SEM . This suOur electron microscopy showed that, despite being much reduced, the haptomonad axoneme still contained some of the canonical non-microtubule components such as the radial spokes, while the PFR was absent . To examThe cells with lateral attachment and unattached cells around the stomodeal valve  had a ce2+ . Thus, our data indicate that there are different regulatory mechanisms for adhesion in different trypanosomatids. To examine whether the removal of Ca2+ from cells that have already attached to the substrate disrupted their attachment, we incubated attached in vitro haptomonad-like cells (formed by differentiation for 24 hr in vitro) in the medium containing 2 mM EGTA for 30 min, and then counted the number of cells that remained attached to the glass , rather than flexible substances such as mucus material. Overall, there has been a dramatic remodelling of the flagellum in the haptomonad. The strong attachment of the cell to the cuticle of the valve is not just mediated by the attachment plaque but is buttressed by the numerous filaments and filament bundles, which connect the attachment interface to the junctional complexes of the FAZ.The thickness of the filaments in the haptomonad flagellum is 8–10 nm, which is similar to the thickness of intermediate filaments or septin filaments in other eukaryotes . In addiLeishmania is generally thought of as the only site of exo/endocytosis, the attachment interface could potentially support these processes and may be important for the release of factors such as chitinase directly onto the cuticle surface of the stomodeal valve.Numerous vesicles and vesicular structures were seen within the flagellum. The disassembly of the flagellum during haptomonad differentiation requires a reduction in the amount of flagellar membrane and these vesicles may represent the mechanism by which this membrane is removed and transported back to the cell body, albeit recognising the potential impediment caused by basal body and associated structures. However, in addition to these larger vesicular structures there were smaller vesicles seen in close proximity to the attachment interface, with one in the process of fusing to or budding from the flagellar membrane at this point. The attachment plaque is a complex structure associated with a specialised membrane domain requiring many different components that need to be delivered to the attachment interface for assembly. It is entirely feasible that these components are packaged into vesicles before delivery to the interface and not without precedence as the assembly of desmosome connections between mammalian cells occurs through the delivery of vesicles loaded with desmosome proteins in a calcium dependent manner . AlternaL. mexicana haptomonad form was non-replicative  promastigotes were grown at 28 °C in M199 medium with 10% foetal calf serum (FCS), 40 mM HEPES-HCl (pH 7.4), 26 mM NaHCO3 and 5 µg/ml haemin. Cells were maintained in logarithmic growth by regular subculturing.Lutzomyia longipalpis were fed through a chick-skin membrane on heat-inactivated sheep blood containing Leishmania mexicana promastigotes from log-phase cultures at a concentration 2×106 cells/ml. Blood-engorged females were separated and maintained at 26 °C and high humidity with free access to a 50% sugar solution, with a 14 hr light/10 hr dark photoperiod. They were dissected on day 10 after a bloodmeal, and the dissected guts were fixed for 24 hr at 4 °C in Karnovsky fixative ), transferred to the washing solution (0.1 M cacodylate buffer with 2.7% glucose) and kept at 4 °C.Sand fly infection was carried out as described in Leishmania infected guts were washed in 0.1 M cacodylate buffer (pH 6.9) and then incubated in 1% osmium tetroxide in 0.1 M cacodylate buffer containing 1.5% potassium ferricyanide, for 1 hr in the dark. The guts were then washed with ddH2O, incubated in freshly prepared 1% thiocarbohydrazide (TCH) for 20 min in the dark, washed in ddH2O, and incubated in 2% osmium tetroxide in ddH2O for 30 min in the dark. The guts were washed again with ddH2O and incubated in 1% uranyl acetate in ddH2O overnight, at 4 °C and in the dark. Then, the guts were washed with ddH2O and dehydrated in an ethanol series , and 2×absolute ethanol; 10 min / step). The guts were embedded in TAAB 812 hard resin . Resin pieces containing the stomodeal valve region of the gut were mounted onto aluminium pins using conductive epoxy resin, sputter coated with a thin (12–14 nm) layer of gold, and then imaged in a Zeiss Merlin VP Compact fitted with a 3view2XP system . Serial images of the block face were recorded at an accelerating voltage of 1.8 kV and an aperture size of 20 μm. The pixel size and the dwell time for image capture were 5 nm and 3 μs, respectively, and the slice thickness was 100 nm. Images were recorded using an OnPoint backscattered electron detector . Data were segmented manually to 3D models, using 3dmod , and collected on formvar-coated slot grids. Sections were stained with Reynolds lead citrate before imaging at 120 kV, on a Jeol JEM-1400Flash  with a OneView  camera. Each individual tomogram was produced from a total of 240 4K x 4K pixel images  acquired automatically using SerialEM . Individhttps://bio3d.colorado.edu/imod/doc/3dmodguide.html). The 3D models of the stomodeal valve or plastic substrate with high electron density outlines were generated using the isosurface function of 3dmod. Also, we deposited the original SBFSEM (EMPIAR-11463 and EMPIAR-11464) and electron tomography (EMPIAR-11467 and EMPIAR-11468) data as an MRC image file, which can be opened and analysed using 3dmod, on EMPIAR (https://www.ebi.ac.uk/empiar/).Three-dimensional models of the SBFSEM and electron tomography data were created by manually tracing structures in each slice, and surface meshes were computed from the traced contours in each slice using 3dmod  which were cut into small pieces of ~5 × 5 mm and sterilised with 100% ethanol (for light microscopy and SEM) or 13 mm round Thermanox plastic coverslips  scratched with sandpaper and sterilised with 100% ethanol (for SBFSEM) in a 24-well plate with 1 ml of M199 medium at 28 °C with 5% CO2 for 24 hr (for light microscopy and SEM) or 72 hr with M199 medium being replaced every 24 hr (for SBFSEM).Axenic haptomonad-like cells were generated by culturing 1×10For light microscopy of living cells, in vitro haptomonad-like cells attached to a piece of a gridded glass coverslip were washed twice in DMEM, incubated in DMEM with Hoechst 33342 (1 µg/ml) for 5 min and then washed twice in DMEM. Coverslip pieces were mounted onto another glass coverslip and then onto a glass slide, with the cell attachment side facing up. Attached cells were imaged using a Zeiss ImagerZ2 microscope with 63×objective and a Hamamatsu Flash 4 camera.6 cells/ml) were cultured in a µ-dish 35 mm, high grid-500 glass bottom  for 12 hr, and the dish was washed five times with fresh M199 medium before the start of imaging. Cells about to adhere to the glass were recorded using Zeiss LSM 880 confocal microscopy with 63×objective for 24 hr at 28 °C with 5% CO2 in a chamber with controlled temperature and CO2 concentration.For time-lapse observation, log phase promastigotes . After an hour, coverslips were washed once in PEME and once in ddH2O. The coverslips were then dehydrated using increasing concentrations of ethanol , and 2×absolute ethanol; 10 min / step). The coverslips were then critical point dried, mounted onto SEM stubs using carbon stickers, and sputter coated with a layer of 12–14 nm of gold. Images were taken on a Hitachi S-3400N scanning electron microscope at 5 kV, at a 5.5 mm working distance.In vitro haptomonad-like cells on a piece of a gridded glass coverslip were fixed with 2.5% glutaraldehyde in PEME  were cultured on ~5 × 5 mm pieces of gridded glass coverslips in a 24-well plate with 1 ml of control M199 medium (complete M199 medium including 10% ddH2O) or M199 media including 10% ddH2O and 1 or 2 mM EGTA  for 24 hr at 28 °C with 5% CO2. The coverslips were washed twice with 1 ml of DMEM, incubated in 1 ml of DMEM with Hoechst 33342 (1 µg/ml) for 5 min, and washed twice with 1 ml of DMEM. The glass pieces were mounted with another glass coverslip on a glass slide. The cells were imaged using a Zeiss ImagerZ2 microscope with 20×objective and Hamamatsu Flash 4 camera.Log phase promastigotes (1×106 cells/ml) were cultured on ~5 × 5 mm pieces of gridded glass coverslips in a 24 well plate with 1 ml of M199 medium for 24 hr at 28 °C (in a 5% CO2 atmosphere). The coverslips were transferred into 1 ml of control M199 medium or M199 medium with 2 mM EGTA, respectively and incubated for 30 min. The coverslips were washed twice with 1 ml of DMEM, incubated in 1 ml of DMEM with Hoechst 33342 (1 µg/ml) for 5 min, and washed twice with 1 ml of DMEM. The glass pieces were mounted with another glass coverslip on a glass slide. The cells were imaged using a Zeiss ImagerZ2 microscope with 20×objective and Hamamatsu Flash 4 camera. To quantify cell adhesion under different conditions, the number of cells attached to a single grid lattice area was counted manually using Fiji (Log phase promastigotes (1×10ing Fiji .6 cells/ml) were cultured on formvar-coated 200 mesh nickel grids in a 6 well plate with 5 ml of M199 medium, for 24 hr at 28 °C (with 5% CO2). In vitro haptomonad-like cells attached on the formvar membrane were treated with 1% IGEPAL in PEME  for 5 min, fixed with 2.5% glutaraldehyde in PEME for 10 min and stained with 1% aurothioglucose in ddH2O. The samples were observed using a Jeol JEM-1400 Flash transmission electron microscope operating at 120 kV and equipped with a OneView 16-megapixel camera .Promastigotes (1×107 cells/ml) were centrifuged at 800 g for 10 min and the cells were resuspended in DMEM. In a 15 ml of Falcon tube containing 2 ml of 40% Ficoll 400  in ddH2O at the bottom, 2 ml of 10% Ficoll 400 in M199 medium without FCS and then 2 ml of the concentrated parasites in DMEM were overlaid. The tube was centrifuged at 1300 g for 10 min and parasites located at the upper 10% Ficoll boundary were collected, centrifuged at 800 g for 5 min and resuspended in complete M199 at a cell concentration of 1×106 cells/ml. Log phase promastigotes and in vitro purified metacyclics  were cultured on gridded glass coverslips in a 24 well plate with 1 ml of complete M199 medium for 24 hr at 28 °C with 5% CO2, and the number of attached cells per grid area was quantified in the same method as described above.In vitro L. mexicana metacyclics were purified as described in t-test carried out with Microsoft Excel. Differences were considered significant at the level of p<0.05. Data were plotted with Microsoft Excel or the Matplotlib package in Python  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Calcium dependency of the attachment to the valve in infected flies should be addressed. In addition, it will also be important to test its effects on transmission by bite. If the authors could provide evidence to support the claim that haptomonads are important to transmission, this would be a great addition to the field.2) Apart from sharing structural elements of the attachment plaque, there is no evidence that the forms that attach in vitro represent an authentic and unique life cycle stage in vivo. If by "haptomonad", the authors are referring to any promastigote that attaches to a surface via the formation of a flagellar attachment plaque, then this should be explained and clarified in the manuscript.Reviewer #1 (Recommendations for the authors):While the studies lend impressive details to a previously described aspect of promastigote colonization of the stomodeal valve, they do not further our understanding of the consequences of this colonization, if any, to the ability of infected flies to transmit infection by bite. The degeneration of the valve tissue, including a detachment of the cuticle lining that was previously described in infected flies , was not observed in the current studies. As damage to the valve, mediated by a parasite-derived chitinase, was proposed to facilitate transmission by bite, some discussion as to why they fail to see any damage is warranted.Is 'haptomonad' a term used to describe any promastigote seen in attachment to the stomodeal valve, regardless of morphology? The attached, yellow-colorized cell shown in Figures1B and F with an elongated cell body and extended axoneme has the appearance of a metacyclic form. Would not large numbers of metacyclic promastigotes be expected to colonize the anterior midgut in close proximity to the valve? This raises the question as to whether there is a dedicated stage that is pre-adapted to attachment. This could be addressed in part using the in vitro system. Will metacyclics purified from culture attach in vitro, and do they remodel their flagellum and assume a haptomonad morphology?The calcium2+ dependency of the attachment was confined to the in vitro model. Can the divalent cations be chelated in vivo, perhaps by including EGTA in the sugar meals to address this important question in infected sandflies?Reviewer #2 (Recommendations for the authors):Line 29-30: haptomonad is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available.Lines 74-75: The literature cited here by the authors provides an overview of promastigotes attachment and flagellar modifications inside the sand fly vector. At Wakid and Bates, 2004, the different surfaces for in vitro attachment were proposed but no haptomonad was specifically implicated or status of differentiation was proposed and only "promastigotes" were termed. It does show the formation of hemidesmossomes in vitro but this is no guarantee that any promastigote can't do it or isn't an artifact from in vitro conditions. Due to that, authors cannot claim with the data provided that it's a protocol for having haptomonads in vitro. The manuscript should be adjusted to harbor accurate discussion and conclusions. How do the authors guarantee the hemidesmosomes found in vitro are comparable to haptomonads in vivo ones?Line 87: Although haptomonads are predicted to have an important role in transmission, no data is available demonstrating its role. Too much to conclude here unless a formal demonstration is provided.Line 138: how do the authors know the valve is heavily infected as no counts or infection status were provided?Lines 145-147: Can this be due to sample prep/processing? The asymmetry?Line 191: Vide comment for lines 74-75.Lines 196-200: How the authors are sure of the structure being a clump of haptomonads? And not just an aggregate of cells in multiplication as normally found in cultures?Line 227: The discussion on this topic should be carefully revised. For example, video 6 shows an in vitro promastigote attaching to the surface. This attachment has a hemidesmosome structure formation that resembles the in vivo haptomonad ones. However, this is no evidence that haptomonad differentiation is happening in vitro.Lines 259-269: This section of the manuscript gives interesting information on the location of RSP4/6 and PFR2 on the attaching promastigotes to the scratched surface. Again, it is not conclusive of a haptomonad differentiation situation. The lack of PRF signal can only be concluded by the data presented as "disassembled during promastigote attachment and hemidesmosome formation". This is important information and I suggest the authors explore the strength of the data.Lines 271-291: Same issue as before. To claim in vitro haptomonad formation the authors will need a more detailed comparison with fly-derived haptomonads. This is extremely challenging to achieve though. However, the lack of material does not allow for in vitro assumptions claimed in the manuscript. The current manuscript will be significantly more accurate if the authors focus only on the data and interesting details about the flagellum attachment to the surface that "can" be similar to the real scenario inside the sand fly.The discussion that follows from here should be adjusted based on the comments above. The data is important and provide details not explored. Claiming it's a haptomonad process in vitro is only damaging the dataset as this cannot be concluded. Also, this manuscript will benefit a lot by having a detailed description of how to analyze and get to the 3D models presented. No detailed description was provided. This had a strong potential for usage beyond the Leishmania/sand fly field.Line 478: Haptomonad is a cryptic life stage with an unknown ontology.How can the authors guarantee the in vitro cells have a similar differentiation pattern to haptomonads found in vivo?Line 487: Any media formulation changes will likely promote drastic changes on parasite growth and differentiation. Why for in vitro live cell imaging the authors changed the culture media conditions?Why no statistics detailed methods part is available? Essential revisions:1) Calcium dependency of the attachment to the valve in infected flies should be addressed. In addition, it will also be important to test its effects on transmission by bite. If the authors could provide evidence to support the claim that haptomonads are important to transmission, this would be a great addition to the field.Leishmania proteins such as GP63 that require calcium and are important for parasite development in the sand fly. However, despite these concerns, we did do this experiment, just in case there was an effect  promastigotes from log-phase cultures (day 3-4) at a concentration 106 cells/ml. Blood-engorged females were separated and maintained at 26°C with free access to sugar solution only or sugar solutions containing 5 mM or 20 mM EGTA; all sugar solution contained blue food colour to be sure that dissected females took the sugar solution. On days 6 and 9 post-infection (PI) females were dissected in drops of saline solution. In total we dissected 42 females fed on sugar only, 13 females fed on sugar with 5 mM EGTA and 41 females fed on sugar with 20 mM EGTA. The individual guts were checked for presence and localization of Leishmania promastigotes under the light and fluorescence microscope Olympus BX51. Special emphasis was given to the colonization of the stomodeal valve. Levels of Leishmania infections were graded into four categories: negative, light (< 100 parasites/gut), moderate (100-1000 parasites/gut) and heavy (>1000 parasites/gut).2) Apart from sharing structural elements of the attachment plaque, there is no evidence that the forms that attach in vitro represent an authentic and unique life cycle stage in vivo. If by "haptomonad", the authors are referring to any promastigote that attaches to a surface via the formation of a flagellar attachment plaque, then this should be explained and clarified in the manuscript.L. braziliensis complex in the hindgut; Killick-Kendrick et al., 1977); therefore, the unifying feature of haptomonads is their morphology and the formation of an attachment plaque. It is for that reason we termed them haptomonad. Given the referees uncertainty and lack of molecular markers we have referred to them throughout the manuscript as in vitro haptomonad-like cells. We have revised the manuscript and added explanations to clarify the definition of the in vitro haptomonad-like cell (Line 84-89 and 509-511).A number of the issues raised by the reviewers focus on the terminology used in this study. The in vitro cultured cells we use to initiate this process are widely regarded as promastigotes. During the process these cells clearly differentiate changing shape and morphology, forming an attachment plaque. in vivo cells exhibiting an attachment plaque were defined as haptomonads . In the intervening time since this term was coined, based solely on morphology and the formation of an attachment plaque, no molecular markers of haptomonads have been defined. Moreover, other aspects of the biology of these forms is not helpful as haptomonads in other species can occur in other places (e.g. Reviewer #1 (Recommendations for the authors):While the studies lend impressive details to a previously described aspect of promastigote colonization of the stomodeal valve, they do not further our understanding of the consequences of this colonization, if any, to the ability of infected flies to transmit infection by bite. The degeneration of the valve tissue, including a detachment of the cuticle lining that was previously described in infected flies , was not observed in the current studies. As damage to the valve, mediated by a parasite-derived chitinase, was proposed to facilitate transmission by bite, some discussion as to why they fail to see any damage is warranted.We thank the reviewer for this comment. In the paper  and our previous paper , a higher infection dose or a longer duration between blood meal and imaging was necessary to see the clear degeneration of the valve tissue. However, based on the reviewer’s comment, we revisited the SBF-SEM data of the stomodeal valve in detail and found clear detachment of the cuticle lining in the areas where a large number of parasites were attached. We have now included the data as “Figure1—figure supplement 3” and added a description (Line 187-190).Is 'haptomonad' a term used to describe any promastigote seen in attachment to the stomodeal valve, regardless of morphology? The attached, yellow-colorized cell shown in Figures1B and F with an elongated cell body and extended axoneme has the appearance of a metacyclic form. Would not large numbers of metacyclic promastigotes be expected to colonize the anterior midgut in close proximity to the valve? This raises the question as to whether there is a dedicated stage that is pre-adapted to attachment. This could be addressed in part using the in vitro system. Will metacyclics purified from culture attach in vitro, and do they remodel their flagellum and assume a haptomonad morphology?In this paper, cells are classified as ‘haptomonads’ if they have (i) a short, wide cell body, (ii) a shortened axoneme, and (iii) attached to the substrate through an electron-dense attachment plaque. We regard other attached cells with an elongated flagellum and axoneme with a small attachment plaque  as promastigote cells either differentiating to or away from the haptomonad form.According to the morphological criteria in the previous studies , the cells we saw either attached to or near the stomodeal valve had either a metacyclic  or leptomonad  morphology . In fact the majority of the cells attached to the valve laterally through the flagellum side or were unattached had a leptomonad morphology.Leishmania culture as described in Späth and Beverley, Exp. Parasitol., 2001, and conducted the in vitro attachment assay. From this experiment, we confirmed that in vitro purified metacyclics had little ability to attach. We have added this data as “Figure 4—figure supplement 3” and descriptions (Line 399-449 and 628-681). The lack of attachment by metacyclic forms suggests those attached cells with a long flagellum are likely leptomonad forms in the process of attachment, which is consistent with the suggestion of Rogers et al., 2002 who described the leptomonad as the precursor of the haptomonad.To address whether metacyclics are able to attach, we purified metacyclics The calcium2+ dependency of the attachment was confined to the in vitro model. Can the divalent cations be chelated in vivo, perhaps by including EGTA in the sugar meals to address this important question in infected sandflies?We appreciate this suggestion; however, as outlined above, we believe the results of this experiment are difficult to interpret and when we fed EGTA to sand flies there was no effect on haptomonad attachment (Author response images 1-3). We believe EGTA is more suited for use in relatively well defined in vitro conditions such as those we used to examine its effect on cell adhesion not in complex biological systems.Reviewer #2 (Recommendations for the authors):Line 29-30: haptomonad is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available.We have revised the text referring to the relationship between transmission and the haptomonad form to clarify that a definitive link between the two has not been demonstrated .Lines 74-75: The literature cited here by the authors provides an overview of promastigotes attachment and flagellar modifications inside the sand fly vector. At Wakid and Bates, 2004, the different surfaces for in vitro attachment were proposed but no haptomonad was specifically implicated or status of differentiation was proposed and only "promastigotes" were termed. It does show the formation of hemidesmossomes in vitro but this is no guarantee that any promastigote can't do it or isn't an artifact from in vitro conditions. Due to that, authors cannot claim with the data provided that it's a protocol for having haptomonads in vitro. The manuscript should be adjusted to harbor accurate discussion and conclusions. How do the authors guarantee the hemidesmosomes found in vitro are comparable to haptomonads in vivo ones?We acknowledge that our approach is unable to confirm at the molecular level that the haptomonad-like cells we generate in vitro are equivalent to those observed in the sand fly. As highlighted by the reviewer we do show that the ultrastructural composition of the attachment plaque complex is almost identical between haptomonads in the sand fly and our in vitro haptomonad-like cells. In addition, other aspects of these cells are comparable such as (i) formation of filamentous structures within the flagellum, (ii) shortened axoneme with central pair microtubules, (iii) disassembled PFR, (iv) expanded FAZ structure, (v) cell body dimensions, and (iv) the formation of the extracellular filamentous gel-like matrix. We consider that these aspects support that the in vitro haptomonad-like cells mimic the haptomonads in vivo.Moreover, given the similarities at the ultrastructural level it would be surprising to find that the proteins within the hemidesmosomes in the sand fly haptomonads differed significantly from those in the in vitro derived haptomonad-like cells and would suggest that the cell can form the same structure in two different ways. However, as the reviewer mentioned, these structural and morphological similarities are not sufficient to ensure that the in vitro haptomonad-like cells are the same as in vivo haptomonads. Therefore, following the reviewer’s suggestion, we have adjusted the manuscript explicitly highlighting the caveats of our approach .Line 87: Although haptomonads are predicted to have an important role in transmission, no data is available demonstrating its role. Too much to conclude here unless a formal demonstration is provided.We revised the text at this point as explained above (Line 113-114).Line 138: how do the authors know the valve is heavily infected as no counts or infection status were provided?We have vast experience in observing heavily infected sand fly gut and stomodeal valve, and quantitatively assessing the intensity of their infection by counting the infected parasites, as we showed in our previous paper . In this instance, the density of the haptomonads in the SBF-SEM slices  is comparable to the light micrographs of the heavily infected stomodeal valve in the previous work . Given the density of the attached haptomonads, we consider that the stomodeal valves used in this study are heavily infected . We have modified the manuscript to explain this (Line 194-196).Lines 145-147: Can this be due to sample prep/processing? The asymmetry?We have previously shown that the anterior cell tip in the promastigote cell has an asymmetric extension along one side of the flagellum  and can be seen in other papers as well .This asymmetry results in the anterior end of the cell body crossing the flagellum at an oblique angle when viewed in certain orientations . Given that this feature has been observed independently, it is unlikely an artefact due to sample preparation and/or processing. We consider this to be an intrinsic cell morphological feature of free-swimming promastigotes, and this asymmetric extension of the anterior cell body was much shorter in haptomonads .Line 191: Vide comment for lines 74-75.We have revised the text as mentioned above (Line 264).Lines 196-200: How the authors are sure of the structure being a clump of haptomonads? And not just an aggregate of cells in multiplication as normally found in cultures?We believe the large clumps of cells observed by SEM attached to the substrate are a mixture of haptomonad-like cells actually attached to the substrate and promastigotes that are not attached to the substrate, which we have now explicitly outlined in the manuscript. The origins of these clumps are not clear –They might result from the continued replication of an attached in vitro haptomonad-like cell, which is able to generate a promastigote daughter cell which remains associated with the attached cell, resulting in a clump after several cell cycles.Or they might result from the attachment of a number of cells within an aggregate found within normal culture.However, whatever their origin when we examine such a cell clump by SBF-SEM as shown in Figure 3B and Figure 3—figure supplement 1, we observed that these clumps contain many haptomonad-like cells with clear attachment plaques.Line 227: The discussion on this topic should be carefully revised. For example, video 6 shows an in vitro promastigote attaching to the surface. This attachment has a hemidesmosome structure formation that resembles the in vivo haptomonad ones. However, this is no evidence that haptomonad differentiation is happening in vitro.We have revised the text describing this experiment (Line 320-386).Lines 259-269: This section of the manuscript gives interesting information on the location of RSP4/6 and PFR2 on the attaching promastigotes to the scratched surface. Again, it is not conclusive of a haptomonad differentiation situation. The lack of PRF signal can only be concluded by the data presented as "disassembled during promastigote attachment and hemidesmosome formation". This is important information and I suggest the authors explore the strength of the data.We thank the reviewer for the comment. We have revised the manuscript to make our description more accurate (Line 387-398). We have analysed these data further by measuring the length of the mNeonGreen::PFR2 signal and mCherry::RSP4/6 signal in in vitro adhering cells . This showed that 30% of mature in vitro haptomonad-like cells (9/30 cells) appeared to lose the mNG::PFR2 signal, but the mCh::RSP4/6 signal was still present in all the cells.Lines 271-291: Same issue as before. To claim in vitro haptomonad formation the authors will need a more detailed comparison with fly-derived haptomonads. This is extremely challenging to achieve though. However, the lack of material does not allow for in vitro assumptions claimed in the manuscript. The current manuscript will be significantly more accurate if the authors focus only on the data and interesting details about the flagellum attachment to the surface that "can" be similar to the real scenario inside the sand fly.We thank the reviewer for the important suggestion. As mentioned above, we have adjusted the text in which describe the relationship between the in vitro haptomonad-like cell and the in vivo haptomonad .The discussion that follows from here should be adjusted based on the comments above. The data is important and provide details not explored. Claiming it's a haptomonad process in vitro is only damaging the dataset as this cannot be concluded. Also, this manuscript will benefit a lot by having a detailed description of how to analyze and get to the 3D models presented. No detailed description was provided. This had a strong potential for usage beyond the Leishmania/sand fly field.We have revised the manuscript based on the reviewer’s suggestions to ensure the caveats of our approach are clear . We have included a detailed description of how to analyse the 3D models (Line 756-763), and have added videos showing a rotated view of each 3D model . In addition, we have deposited the SBF-SEM and tomography data on the EMPIAR, so that everyone can access the data (Line 763-766).Line 478: Haptomonad is a cryptic life stage with an unknown ontology.How can the authors guarantee the in vitro cells have a similar differentiation pattern to haptomonads found in vivo?As the reviewer suggests, we still need a formal demonstration to guarantee that the in vitro cells have a similar differentiation pattern to in vivo haptomonads. An important step to guarantee this would be to identify molecular markers of haptomonads in the sand fly. However, we agree with the reviewer that, without molecular markers, these data of in vitro haptomonad-like cells adhering may not closely align to the differentiation pattern of haptomonads inside the sand fly. We therefore revised the descriptions in relation to this .Line 487: Any media formulation changes will likely promote drastic changes on parasite growth and differentiation. Why for in vitro live cell imaging the authors changed the culture media conditions?Leishmania in previous studies , and we ourselves have confirmed that DMEM did not affect cell morphology or protein localisation in short-time observations. However, as we indicated in Materials and methods, in the time-lapse observation, we washed cells with fresh M199 medium and the differentiation of cells was observed in M199 medium. We have adjusted the text to make this clearer (Line 777-778).We routinely wash cells in DMEM without phenol red and FCS in preparation for live cell light microscopy , as the FCS stops the cells becoming immobilised on the glass surface and the phenol red causes an increase the level of background fluorescence in the red channel. DMEM has been used to wash Why no statistics detailed methods part is available?We have now added a statistics section to the Materials and methods (Line 864-868).


Metarhizium fungi have multiple lifestyles and remarkable stress tolerance. Some species, especially M. robertsii, are emerging models for investigating the mechanisms underlying ecological adaptation in fungi. Here we review recently identified new downstream branches of the MAPK cascades in M. robertsii, which controls asexual production (conidiation), insect infection and selection of carbon and nitrogen nutrients. The Myb transcription factor RNS1 appears to be a central regulator that channels information from the Fus3- and Slt2-MAPK cascade to activate insect infection and conidiation, respectively. Another hub regulator is the transcription factor AFTF1 that transduces signals from the Fus3-MAPK and the membrane protein Mr-OPY2 for optimal formation of the infection structures on the host cuticle. Homologs of these newly identified regulators are found in other Metarhizium species and many non-Metarhizium fungi, indicating that these new downstream signaling branches of the MAPK cascades could be widespread.Fungi rely on major signaling pathways such as the MAPK (Mitogen-Activated Protein Kinase) signaling pathways to regulate their responses to fluctuating environmental conditions, which is vital for fungi to persist in the environment. The cosmopolitan  Metarhizium is a genus in the Clavicipitaceae family, and it represents a continuum of species and strains that are extraordinarily versatile , and the insect is killed by a combination of fungal growth and toxins. Finally, the mycelium grown in the hemocoel reemerges from the dead insect, resulting in a muscardine cadaver covered with dark green conidia. The formation of appressorium by Metarhizium is similar to plant pathogenic fungi such as Magnaporthe oryzae and Colletotrichum lagenarium , Fus3-, Hog1-, and Slt2-MAPK cascades are all identified  is also necessary for appressorium formation and following cuticle penetration, and is not involved in hemocoel colonization  and is conserved in the Ascomycete fungi. The Pks1 gene cluster for the conidial pigmentation is also controlled by the Hog1-MAPK via the central regulatory pathway  strain with the MAPK mutants using RNA-Seq analysis revealed many genes regulated by the MAPKs in Aftf1, and to a lesser extent the strain Aftf1OE with Aftf1 overexpressed was delayed, and their pathogenicity was accordingly reduced. Therefore, a precisely modulated optimal level of the AFTF1 protein is needed to choreograph appressorial formation. The expression of Aftf1 is induced by the transcription factor STE12 (named as MrSt12 in M. robertsii); MrSt12 achieves this by physically interacting with the cis-acting element (ATGAAACA) in the promoter of Aftf1. As with the transcription factor STE12 in many other fungi, the activity of MrSt12 is directly phosphorylated by the Fus3-MAPK for activation. In M. robertsii, the deletion mutant of MrSt12 is unbale to form appressorium and is thus nonpathogenic, suggesting that MrSt12 also regulates genes that are not controlled by AFTF1  and M. oryze (XP_003715433), suggesting that the Fus3-MAPK/STE12/AFTF1 signaling branch could be widespread.During cuticle penetration, the transcription factor AFTF1 with a Zn2Cys6 fungal type DNA binding domain is positively regulated by the Fus3-MAPK cacade. Compared to the WT strain, appressorial formation by the deletion mutant of Aftf1 expression to ensure that the expression level of Aftf1 is not too high for optimum effect  in the OPY2 protein were found to interact with STE50; CR-A binds to STE50 constitutively to transmit signals to both the Hog1- and Fus3-MAPK cascade, while CR-B binds to STE50 under glucose-rich conditions to transmit the signal preferentially to the Hog1-MAPK cascade  is also directly regulated by the Fus3-MAPK cascade, but shows no direct relationship with the transcription factor AFTF1  that are not derived from insects. This is repressed by favored organic carbon and nitrogen nutrients, including glucose and glutamine. Therefore, the Fus3-MAPK/RNS1 branch controls fungal nitrogen and carbon metabolism and is implicated in the entomopathogenicity of M. robertsii, two other regulatory mechanisms have been widely documented in fungi, which ensure that the genes for utilization of less-favored carbon or nitrogen sources are expressed only in the absence of the favored nutrients so that fungi preferentially utilize favored carbon  and nitrogen  sources. One mechanism is the nitrogen metabolite repression (NMR). The regulatory protein NmrA deactivates the GATA transcription factor AREA in the presence of favored nitrogen, whereas in the absence of favored nitrogen sources, NAD+ binds to NmrA, which in turn dissociates from AREA, thereby allowing AREA to activate expression of the genes for utilizing less-favored nitrogen sources  are in the 5′ UTR of BrlAβ, which could inhibit the translation of the downstream major ORF (Guo et al., BrlA gene and induces the expression of the transcript BlrAα. Since no uORFs are in the 5′ UTR of BrlAα, the major ORF can be highly translated. The BrlA protein in turn activates the expression of AbaA and WetA for optimal conidiation (Meng et al., As described above, the BrlA cannot differentiate to produce conidiophores (Zeng et al., Rns1 can produce conidia though its conidial yield is reduced, suggesting that RNS1 only regulates the expression of the transcript BrlAα and the transcript BrlAβ could also function during conidiation. The contributions of BrlAα and BrlAβ to conidiation remain to be clarified. In addition to conidiation, deletion of the gene encoding Slt2-MAPK also reduced the tolerance to hyperosmotic stress and to the cell wall-disturbing agent Congo red; but deletion of Rns1 had no impact on the tolerance of M. robertsii to hyperosmotic stress and cell wall-disturbing agent. Therefore, the Slt2-MAPK cascade has other downstream branches than the Slt2-MAPK/RNS1.The aerial hyphae of the deletion mutant of Metarhizium fungi have multiple lifestyles and remarkable stress tolerance (Słaba et al., M. robertsii is an emerging model to investigate the mechanisms underlying ecological adaptation in fungi. Major signaling pathways such as the conserved MAPK cascades play key roles in fungal sense and response to fluctuating environmental conditions, which is vital for fungi to persist in environment. In recent years, several novel downstream signaling branches of the MAPK cascades have been discovered in M. robertsii, which regulate conidiation and nutrient selection and entomopathogenicity (Metarhizium species and many non-Metarhizium fungi, indicating that these new downstream signaling branches of the MAPK cascades could be widespread.The genicity . The MybWF conceived the idea and wrote the manuscript. DT and XT wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China Grants 31872021 and 32172470.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


White cord syndrome (WCS) shows high intramedullary signaling in T2-weighted MRI with worsening motor nerve symptoms after cervical spinal decompression surgery. It has been reported in only 13 cases.An 81-year-old man had numbness, weakness, and impaired fine motor control in both upper limbs for the previous five years. C3, C4, C6, open-door laminoplasty, and C5 laminectomy were performed. Intraoperative transcranial motor evoked potential normalization by compound muscle action potential showed an 80% reduction in amplitude in the right abductor pollicis brevis and a 96% reduction in the right abductor hallucis. Tetraplegia occurred immediately after the operation. Magnetic resonance imaging (MRI) on the day after the operation showed intramedullary T2 high signals at the C4 and C5 levels. According to Brunnstrom's staging, the upper and lower right limbs and the lower left limb were at stage two, and the upper left limb was at stage three, six months after the operation. Thirteen cases of WCS have been reported in the literature. These were thought to be caused by reperfusion due to decompression. White code syndrome (WCS) is a very rare postoperative cervical complication, with only 13 reported cases at the time of writing. ..14].Strong quadriplegia was recognized, especially on the right side, which was consistent with the findings of MEP monitoring immediately after the operation. The decompression on CT was sufficient, and the follow-up consisted of only steroid administration. An MRI performed the day after the surgery revealed marked intramedullary T2-high signals at the C4 and C5 levels Figure .The postoperative JOA score was -0.5 points. He was transferred to a recovery-phase rehabilitation hospital and discharged to his home six months later, but the Brunnstrom stage was two for the upper right and lower left limbs and three for the upper left limb.WCS was first reported in 2013; six cases were reported in 2020, and a total of only 13 cases have been described overall  became worse after six months . EspeciaWCS is thought to cause edema and bleeding due to reperfusion syndrome as a result of rapid decompression ,16. HypeA newly developed neurological deficit that may be attributed to an intraoperative iatrogenic cord injury is not WCS. Acharya et al. reported a case of quadriplegia following cervical posterior decompression surgery that had been caused by an intraoperative iatrogenic spinal cord injury, and WCS was misdiagnosed . WCS is MEP monitoring under CMAP normalization after peripheral nerve stimulation had a sensitivity of 100% in spinal surgery and seemed to be very useful for predicting postoperative motor paralysis such as WCS during an operation ,16. HoweIn the case described here, the preoperative JOA score was more than 10 points. The risk of developing WCS may be higher in cases where the neurological symptoms are mild, even if the spinal cord is under strong compression in the imaging. The operation indication must be carefully judged, and sudden decompression in the operation must be avoided, especially during a posterior approach in cases where the neurological symptoms are mild even if the compression on the image is strong, as in the present cases.WCS has been reported in 14 cases, including our case. WCS is caused by reperfusion, and it seems to be important to avoid rapid decompression in cases with advanced cervical cord compression.


BackgroundOP-29 is a Centers for Medicaid and Medicare Services (CMS) measure to ensure that endoscopists recommend appropriate follow-up intervals after normal colonoscopy in average risk patients. Failure to report OP-29 compliance can adversely affect hospital quality star rating as well as reimbursement for health care. The aim of our quality improvement project was to improve OP-29 compliance to the top decile over three years.MethodologyOur sample included patients between 50-75 years of age who received average risk screening colonoscopies with normal findings. We provided intensive education to endoscopists about the importance of OP-29 compliance, developed an Epic Smartlist that directs our endoscopists to list an appropriate reason for colonoscopy intervals other than 10 years, and monitored OP-29 compliance monthly. We became the first health network in the United States to implement the Lumens endoscopy report writing software  and added the OP-29-related Epic Smartlist to the Lumens colonoscopy note template. All statistical analyses were conducted in SPSS version 26  to compute the means and frequencies of outcomes.ResultsOur sample included 2,171 patients with a mean age of 60.5 years of whom the majority were female (57.2%) and Caucasians (90%). Our OP-29 score increased from 87.47% to 100% over the course of three years, and this steady improvement was seen broadly across our network. We compared our network score averages to our state and national averages and consistently demonstrated higher compliance rates while reaching the top decile by 2020.ConclusionWe believe our improved OP-29 compliance has reduced colonoscopy overutilization, improved health care quality, and reduced health care costs for our patients and health network. To our knowledge, this is the first reported project towards improving OP-29 compliance utilizing the Epic Lumens software. Epic Lumens  added this Smartlist as quick buttons in the standard colonoscopy procedure note templates they built for other organizations to improve health care quality and cost nationally. The Hospital Outpatient Quality Reporting (OQR) Program is a pay-for-quality data reporting initiative that was mandated by the Tax Relief and Health Care Act of 2006 . The CenPayments for hospitals participating in the Hospital OQR Program are determined by specific measures, which identify processes of care, imaging efficiency patterns, care transitions, and patient safety . CMS hasThis article was previously presented as a meeting abstract at the 2021 Digestive Disease Week (DDW) Annual Scientific Meeting on May 21, 2021 .The study took place from January 2017 to March 2020 within our 14-hospital network located in Eastern Pennsylvania and New Jersey. We only included data from the seven hospitals that were part of the network when the study began in 2017. The study was approved by the St. Luke's University Health Network Institutional Review Board (#2020-111). We included all patients between 50-75 years of age who received average-risk screening colonoscopies with normal findings according to the United States Multi-Society Task Force for Colorectal Cancer guidelines. We excluded patients given a follow-up interval of 10 years and those given a follow-up interval other than 10 years for whom a reason was given, such as no adenoma with age > 66 years; life expectancy < 10 years; personal/family history of colon polyps or colon cancer; or comorbidities significantly increasing complication risk from further procedures.This was a prospective quality-improvement initiative that sought to improve OP-29 compliance through various interventions following the Plan-Do-Study-Act model. We first obtained the baseline OP-29 scores, identified barriers to compliance, and provided intensive education to endoscopists about the importance of OP-29 during department meetings. We then developed and implemented an Epic Smartlist that directed endoscopists to recommend a repeat colonoscopy interval and list an appropriate reason if the interval is not 10 years  to compute the means and frequencies of outcomes. Our sample included 2,171 patients with a mean age of 60.5 years of whom the majority were female (57.2%) and Caucasians (90%). Our quality improvement project spanned the course of three years with analysis of OP-29 compliance conducted from January 2017 Q1 to March 2020 Q1. Our OP-29 score increased from 87.47% in 2017 Q1 to 100% in 2020 Q1 Figure . We alsoEndoscopists are expected to provide a follow-up recommendation after a colonoscopy, which is an extremely common clinical scenario requiring guidance . The UniNumerous studies have identified the overuse of colonoscopy as measured by physician recommendations -11. AddiHealth information technology (HIT) has become fundamental to healthcare and is a primary strategy for not only improving the quality of care and optimizing patient management but also enhancing the capability to perform large-scale research and identify effective interventions -14. CommEpic Lumens is the reporting module for endoscopy, which provides tools for viewing and managing endoscopy images sent to Epic by external endoscopy systems . To our Throughout the course of our quality improvement project, we observed a few ongoing challenges. Our clinical analytics team discovered that not all endoscopists were using the Epic Smartlist. Additionally, some endoscopists were not using the Epic Smartlist for all colonoscopies. Given that the endoscopy reporting software was a new addition during our study, this nonadherence could be attributed to the natural difficulties that arise when adjusting to a new tool. These difficulties include learning how to utilize the Smartlist and incorporating it into one’s typical EHR workflow. Another explanation could be that the endoscopists were simply unaware of the Smartlist or were forgetting to utilize it. We also observed that compliance was lower among the non-gastroenterologists and non-employed SLUHN endoscopists. We suspect the lower compliance rate among these groups was due to the limited reach of our education regarding OP-29. The non-gastroenterologists and non-employed SLUHN endoscopists were not a primary target for our intensive education, and thus, were not as informed as our SLUHN endoscopists. We addressed these adherence challenges through additional education and by adding the Epic Smartlist to the Epic Lumens colonoscopy template. This increased our OP-29 compliance rate to 100%, but we anticipate continued intermittent non-adherence and plan to continue to provide regular feedback and training.Our study demonstrates that EHRs possess the capability to improve the quality of care and safety in health care, and these advancements can occur once providers understand and adapt to the new functions consistently and effectively. Our project sought to improve OP-29 compliance at SLUHN over the course of three years. We observed a successful improvement with compliance scores increasing from 87.47% in 2017 to 100% by the end of our study in 2020. We believe our improved OP-29 compliance has led to reduced colonoscopy overutilization, improved healthcare quality, and reduced health care costs for our patients and our health network. Our top decile OP-29 compliance is one of many factors that has contributed to our attainment of a five-star hospital quality rating.


What is the association of nirmatrelvir and ritonavir exposure with the risk of death or COVID-19–related hospitalization when accounting for patient vulnerability to complications from COVID-19 infection?In this cohort study of 6866 individuals with COVID-19, treatment with nirmatrelvir and ritonavir was associated with lower risk of death or hospitalization in the most clinically extremely vulnerable individuals but not in less vulnerable individuals. Individuals who were not extremely vulnerable to experiencing complications from COVID-19, whose median age was 79 years, had greater risk of the outcome while receiving nirmatrelvir and ritonavir, but the finding was not statistically significant.In this study, treatment with nirmatrelvir and ritonavir was not associated with reduced risk of death or hospitalization among individuals who were not extremely vulnerable to complications from COVID-19 infection, regardless of age. This cohort study examines the association of nirmatrelvir and ritonavir with prevention of death or admission to hospital in individuals with different risks of complications from COVID-19 infection. Postmarket analysis of individuals who receive nirmatrelvir and ritonavir (Paxlovid [Pfizer]) is essential because they differ substantially from individuals included in published clinical trials.To examine the association of nirmatrelvir and ritonavir with prevention of death or admission to hospital in individuals with different risks of complications from COVID-19 infection.This is a cohort study of adult patients in British Columbia, Canada, between February 1, 2022, and February 3, 2023. Patients were eligible if they belonged to 1 of 4 higher-risk groups of individuals who received priority for COVID-19 vaccination. Two groups included clinically extremely vulnerable (CEV) people who were severely (CEV1) or moderately immunocompromised (CEV2). CEV3 individuals were not immunocompromised but had medical conditions associated with a high risk for complications from COVID-19. A fourth expanded eligibility (EXEL) group was added to allow wider access to nirmatrelvir and ritonavir for certain other higher-risk individuals who were not in a CEV group, such as those older than 70 years who were unvaccinated.Patients with COVID-19 who received nirmatrelvir and ritonavir were matched to patients in the same vulnerability group; who were of the same sex, age, and propensity score for nirmatrelvir and ritonavir treatment; and who were also infected within 1 month of the individual treated with nirmatrelvir and ritonavir.The primary outcome was death from any cause or emergency hospitalization with COVID-19 within 28 days.There were 6866 individuals included in the study, of whom 3888 (56.6%) were female and whose median (IQR) age was 70 (57-80) years. Compared with unexposed controls, treatment with nirmatrelvir and ritonavir was associated with statistically significant relative reductions in the primary outcome in the CEV1 group  and the CEV2 group . In the CEV3 group, the RD was −1.3%, but the findings were not statistically significant . In the EXEL group, treatment was associated with higher risk of the outcome , but the findings were not statistically significant .In this cohort study of 6866 individuals in British Columbia, nirmatrelvir and ritonavir treatment was associated with reduced risk of COVID-19 hospitalization or death in CEV individuals, with the greatest benefit observed in severely immunocompromised individuals. No reduction in the primary outcome was observed in lower-risk individuals, including those aged 70 years or older without serious comorbidities. Nirmatrelvir and ritonavir reduced the primary composite end point of 28-day risk of death or COVID-19–related hospitalization by 5.6% absolute (88% relative) compared with placebo. By day 28, there were 0 deaths in the nirmatrelvir and ritonavir group and 12 in the placebo group. EPIC-HR reported numerically fewer serious adverse events but more suspected drug-related adverse events with nirmatrelvir and ritonavir than placebo.Nirmatrelvir and ritonavir (Paxlovid [Pfizer]) is an oral antiviral drug combination that targets a key SARS-CoV-2 protease enzyme. Nirmatrelvir and ritonavir was approved based on interim efficacy and safety data from the Evaluation of Inhibition for COVID-19 in High-Risk Patients (EPIC-HR) trial, conducted before the emergence of the Omicron variant.4 The manufacturer announced the closure of the trial in a June 14, 2022, media release, “due to a very low rate of hospitalization or death observed in the standard-risk patient population.”4 Nirmatrelvir and ritonavir was not associated with reduced symptoms (the primary end point), and the media release reported a non–statistically significant reduction of 0.9% absolute (51% relative) in hospitalization or death. Peer-reviewed observational analysis has also shown COVID-19–related hospitalization or death from any cause was significantly lower with nirmatrelvir and ritonavir treatment,5 although by approximately half the magnitude reported in the EPIC-HR trial. Observational studies have demonstrated a benefit of nirmatrelvir and ritonavir treatment in higher risk individuals.6Parallel to EPIC-HR, a further 1141 lower-risk adults were studied in a second trial known as EPIC-SR .7 As in the EPIC-HR trial, we sought to analyze the 28-day risk of death or COVID-19–associated hospitalization in the 4 groups of vulnerable individuals in BC with elevated risk of complications who were given access to nirmatrelvir and ritonavir.Protective associations with nirmatrelvir and ritonavir therapy have been shown to be variable across the different populations and study periods, which suggests the benefit risk profile of nirmatrelvir and ritonavir depends on vulnerability to complications from COVID-19 infection. The benefit-harm profile of nirmatrelvir and ritonavir thus remains uncertain. British Columbia (BC) provides a natural experiment for examining nirmatrelvir and ritonavir according to vulnerability to complications from COVID-19. BC adopted eligibility criteria for nirmatrelvir and ritonavir that differed substantially from participants in the EPIC-HR trial and included individuals who were more comparable with those studied in the EPIC-SR trial. Furthermore, patients enrolled in EPIC-HR were unvaccinated, had no natural immunity from prior COVID-19 infection, were infected by COVID-19 variants that were different from those now circulating, and were not taking drugs with known CYP 3A4 interactions.STROBE) reporting guideline. Informed consent from study participants was not required because all data were deidentified and the study satisfied the minimal risk category for ethics review at the University of British Columbia.We undertook a retrospective cohort study between February 1, 2022, and February 3, 2023, of individuals who had increased vulnerability to complications from COVID-19 infection. Death from any cause and COVID-19–related hospitalization were compared between individuals who were either prescribed or not prescribed nirmatrelvir and ritonavir. The study used anonymized, individual-level, and linkable administrative health databases from the BC Ministry of Health. Prescription data for nirmatrelvir and ritonavir and other drugs were obtained from the PharmaNet database of all prescriptions filled at community pharmacies. COVID-19 vaccination records and polymerase chain reaction (PCR) test records were also obtained from the Ministry of Health. Demographic, diagnostic, and medical procedure data, used to identify clinically extremely vulnerable (CEV) cohorts and perform statistical adjustment, were obtained from the Medical Services Plan database, the hospital Discharge Abstract Database, and the National Ambulatory Care Reporting System database (NACRS). This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , and 1 expanded eligibility (EXEL) population. Two CEV populations included individuals 18 years of age and older who were either severely (CEV1) or moderately (CEV2) immunocompromised. Individuals in the CEV3 population were not immunocompromised but had medical conditions thought to place them at higher than typical risk for complications from COVID-19 infection. These conditions included severe respiratory disorders; certain blood disorders, metabolic disorders, and cancers not captured in the other CEV groups; or insulin-dependent diabetes. A fourth group, the EXEL population, was added on March 17, 2022, to allow wider access to nirmatrelvir and ritonavir for individuals at lower risk of COVID-19–related complications than CEV patients, but who had risk factors that put them at higher risk of complications compared with the general population. Eligibility details for this category are publicly available from the BC Centre for Disease Control.8 Briefly, the EXEL category was focused on older individuals with certain comorbidities who were unvaccinated or undervaccinated according to the Strong Recommendations by the National Advisory Committee on Immunization.9 Individuals 70 years of age or older who were unvaccinated could qualify for nirmatrelvir and ritonavir under the EXEL category. Individuals 70 years of age or older who were fully vaccinated but had a serious medical condition not captured in a CEV category could also qualify. Prescribing physicians were allowed to use discretion in their determination of serious comorbidities.Four mutually exclusive populations of individuals at higher risk for complications from COVID-19 infection were eligible for nirmatrelvir and ritonavir during the study period. Detailed definitions used to identify these populations are provided in eAppendix 1 in Cohorts of individuals with COVID-19, identified from either a PCR test, a prescription for nrirmatrelvir and ritonavir, or both, were assembled from the 4 vulnerable populations. Individuals were not allowed to enter a cohort more than once. For individuals who at different times qualified as nirmatrelvir and ritonavir exposed and unexposed, the nirmatrelvir and ritonavir instance was included. Prior to initiating treatment with nirmatrelvir and ritonavir or having a positive COVID-19 test, individuals required at least 730 days of continuous enrollment in the BC Medical Services Plan (gaps of ≤30 days were permitted). Individuals were excluded from the study if, at the time before their test sample collection date (or 3 days prior to initiating nirmatrelvir and ritonavir if there was no test), they were younger than 18 years of age, had a history of severe kidney or liver disease, pregnancy, use of remdesivir, or, within 30 days before their test, were in hospital for any reason. Detailed exclusion criteria are provided in eAppendix 2 in 10 The HDPS algorithm empirically selected covariates from the 730-day period prior to the COVID-19 test sample collection date. Within each cohort, nirmatrelvir and ritonavir–exposed individuals were matched 1-to-1 with non–nirmatrelvir and ritonavir–exposed individuals, without replacement, using the nearest neighbor method. Parameters used for matching were age (±2 years), sex, propensity score (±0.2 multiplied by the standard deviation of the pooled standard deviation of the logit of the propensity score),11 and year and calendar month (±1 month) of COVID-19–positive test.Individuals prescribed nirmatrelvir and ritonavir were matched to individuals with COVID-19 who were not prescribed nirmatrelvir and ritonavir. Nirmatrelvir and ritonavir–exposed individuals were not required to have a positive PCR test, but all non–nirmatrelvir and ritonavir–exposed individuals required a positive test. Before matching, to minimize potential confounding, high-dimensional propensity score (HDPS) models were estimated for each of the 4 groups.Follow-up for study outcomes in an nirmatrelvir and ritonavir–exposed individual began on their nirmatrelvir and ritonavir initiation date. For each unexposed matched control participant, to avoid immortal time bias, follow-up began on the individual’s COVID-19 test collection date, plus the number of days that lapsed between the test collection date and treatment initiation in the control individual’s nirmatrelvir and ritonavir–exposed counterpart. Where the nirmatrelvir and ritonavir–exposed individual in a match did not have a PCR test, follow-up in the non–nirmatrelvir and ritonavir–exposed individual began 3 days after their PCR test sample collection date.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, codes U07.1 and U07.2) and subsequent admission to hospital. To assess the reliability of this approach, we compared complete hospital discharge records with NACRS records in the year before our study. From March 1, 2021, to February 28, 2022, a NACRS record that indicated subsequent admission to hospital was 88.5% sensitive and 92.3% specific for the presence of an emergency hospital admission record in the hospital discharge abstract database.The primary outcome was a composite outcome of COVID-19–related emergency hospital visit or admission, or death from any cause, within 28 days of an individual’s cohort entry date. A secondary outcome was emergency department visit for any reason, with or without a subsequent admission to hospital. Estimation of hospitalization outcomes relied on emergency department (ED) records after March 31, 2022. This was because complete hospital discharge abstract data for the remainder of study period will not be available until late 2023. Instead, COVID-19–related emergency admissions after March 31, 2022, were identified using ED visit records from the NACRS database, which was complete for our study period. A NACRS record was counted as a COVID-19–related emergency hospitalization if it indicated COVID-19 infection . We used a 95% CI that excluded the null as our level of statistical significance.After assembling nirmatrelvir and ritonavir exposure and outcome data for the matched cohorts in 2 × 2 tables, we estimated the 28-day risk difference (RD) and the 28-day relative risk (RR) for each study outcome. These parsimonious analyses allowed for the straightforward reporting of RDs and numbers-needed-to-treat but did not account for the possible influence of competing risks. The reasonability of this approach was checked by comparing the RR estimates with hazard ratios estimated using the method of Fine and GrayThere were 6866 individuals included in the study, of whom 3888 (56.6%) were female and whose median (IQR) age was 70 (57-80) years. Characteristics of the matched vulnerability groups are shown in Results for the association between nirmatrelvir and ritonavir and risk of death or COVID-19–related emergency hospitalization, the primary end point, are shown in In In the main analysis, nirmatrelvir and ritonavir–exposed individuals did not require a PCR test because COVID-19 was the only indication for nirmatrelvir and ritonavir. A sensitivity analysis that required all nirmatrelvir and ritonavir–exposed individuals to also have a positive PCR test resulted in a subgroup of 1362 individuals from the original 6866 study population. A small number of events prevented reporting the sensitivity analysis in the CEV1 group. In 480 individuals in the CEV2 group, the estimated RD was the same as in the main analysis at −1.7% . In 352 individuals in the CEV2 group, the RD was −1.1% , compared with −1.3% in the main analysis. In 470 individuals in the EXEL group, the RD was 3.4% , compared with 1.0% in the main analysis.2 where a statistically significant reduction of hospitalization or death was seen. The results in the lower-risk EXEL group appeared comparable with the unpublished results of the EPIC-SR trial,4 where no difference in the primary outcome was seen, although we observed a nominally higher hospitalization and mortality rate in those exposed to nirmatrelvir and ritonavir. With one important exception, our results were generally consistent with observational studies of nirmatrelvir and ritonavir,6 where the benefit of nirmatrelvir and ritonavir is driven by high-risk groups within an exposed population. The important exception in our study was that, owing to the stratified approach in our analysis, older age (≥70 years) did not have a statistically significant beneficial association with nirmatrelvir and ritonavir after accounting for other comorbidities.In this observational study of 6866 individuals in BC, nirmatrelvir and ritonavir exposure was associated with a lower risk of death or COVID-19–related hospitalization in individuals with extreme vulnerability to complications from COVID-19. The same association was not observed in lower-risk individuals in the EXEL group, a result that was robust across sex and older vs younger age. The results from our CEV cohorts were compatible with the results of the EPIC-HR trial,Our study has several important strengths. First, nirmatrelvir and ritonavir was assessed in stratified analyses in cohorts that were based on different degrees of vulnerability to COVID-19–related morbidity and in secondary analyses of age and sex. This approach prevented overly parsimonious inferences in the presence of effect estimate modification by these factors. If all vulnerability groups had been combined into one cohort, the RD for the primary outcome in the subgroup of individuals aged 70 years or older would have been −1.4% and statistically significant. However, our analysis showed that individuals aged 70 years or older of age in the EXEL group did not have their risk of the outcome reduced by nirmatrelvir and ritonavir, which is an important finding that could prevent overtreatment with nirmatrelvir and ritonavir in older people who are not extremely vulnerable to complications from COVID-19. Second, use of reference groups with similar COVID-19 vulnerability and who tested positive for COVID-19 close to the time of their matched nirmatrelvir and ritonavir–exposed counterpart avoided confounding by indication and by COVID-19 variant. Third, use of HDPS in the matching algorithms allowed for control of empirical residual confounding by a constellation of factors that could not have been achieved by means of statistical adjustment, or by weighting, because events were sufficiently rare to have limited the number of confounding variables that could have been accommodated with those analytical approaches.Several limitations of our analyses merit mention. Omicron was the main circulating variant of COVID-19 during the study period, and our results may therefore not be applicable to other variants, past or future. Omicron is associated with less severe disease than the Delta variant, which was prevalent during the EPIC-HR trial era. This could be one reason the estimates in our study were attenuated by comparison. Our analyses relied on administrative claims data that may not have captured all factors used by physicians in choosing treatment with nirmatrelvir and ritonavir. To limit the influence from this possible source of bias, we excluded nirmatrelvir and ritonavir–exposed individuals and other individuals with COVID-19 who did not have data indicating which of the 4 vulnerability groups they belonged to. We then analyzed study outcomes stratified by these groups. Future research might focus on creating an approach to assessing COVID-19 vulnerability that is more granular than the 4 vulnerability groups used in this study. Such an approach has the prospect of offering more targeted treatment with nirmatrelvir and ritonavir. More individuals in the nirmatrelvir and ritonavir–exposed group of the EXEL group were unvaccinated (26.6% vs 21.5%). The RD for the primary outcome in the EXEL group was 1.0%, which equates to an RR of 1.30. Even if the independent RR of the primary outcome in nonvaccinated vs vaccinated non–nirmatrelvir and ritonavir–exposed individuals in the EXEL group was an un unrealistic RR of 100, the RR for the primary outcome in the EXEL group in nirmatrelvir and ritonavir–exposed vs unexposed individuals would have been at most 1.23. Thus, the imbalance in vaccination cannot account for the reported association. Our cohort criteria required a positive COVID-19 PCR test. This may have limited the generalizability of our findings.In this cohort study of individuals with COVID-19 in BC between February 1, 2022, and February 3, 2023, use of nirmatrelvir and ritonavir appeared to reduce the risk of the composite outcome of death or COVID-19–related hospitalization in high-risk, mostly moderately or severely immunocompromised individuals. Stratified analysis of individuals according to vulnerability to complications from COVID-19 is crucial to understanding which individuals should use nirmatrelvir and ritonavir. In our study, lower-risk individuals, including those older than 70 years who were not moderately or severely immunocompromised, did not appear to benefit from nirmatrelvir and ritonavir.