Uterine leiomyomas are common benign neoplasms. While generally easily diagnosed, variations in size and type of degeneration can drastically alter the imaging findings and result in diagnostic uncertainty. We present the case of a 34-year-old female with a large, cystic, pelvi-abdominal mass that was discovered in advanced pregnancy and misdiagnosed as an ovarian tumour. Intra-uterine demise of a viable foetus during an induction of labour and clinical deterioration necessitated further imaging and emergent surgical intervention. Further imaging, intra-operative findings and histopathological correlation revealed a pedunculated uterine leiomyoma with hyaline degeneration and areas of necrosis, complicated by left ovarian vein thrombosis. In addition to the intra-uterine demise, the unfortunate outcome included a total abdominal hysterectomy and unilateral salpingo-oophorectomy in a young patient. Typical leiomyomas are easily detected with imaging, however the varied clinical presentation and imaging findings in degenerating leiomyomas can prove a diagnostic challenge. This case describes a rare manifestation of a common condition and its subsequent diagnostic dilemma resulting in dire foeto-maternal outcomes.Leiomyoma; Hypercalcaemia; Ovarian Vein Thrombosis; Pregnancy; Intra-Uterine Foetal Demise. Simultaneous heterogeneity amongst the patient profile  further complicates accurate analysis.3 As a result, the relationship between leiomyomas and adverse obstetric outcomes is incompletely understood.The current literature suggests that the majority of women with uterine fibroids will have normal pregnancy outcomes.3 Local advances in reproductive health, as well as the current trend of delaying childbearing, further increase the relevance to the South African population, as treating clinicians can expect to see more women with leiomyomas presenting for antenatal and obstetric care.5Accurate diagnosis of leiomyomas in pregnancy is particularly important in a South African context as an increased prevalence of leiomyomas in the African population was described by Stewart et al.Typical leiomyomas are readily diagnosed radiologically; however, the appearance of uterine leiomyomas is commonly altered by degenerative changes including cystic, hyaline, myxoid and red degeneration. This can make differentiation between leiomyomas and other adnexal or other intra-abdominal pathology challenging.Ultrasound is used to diagnose and monitor the growth of leiomyomas, and typically shows a well-defined hypoechoic mass. Intra-lesional cystic areas or calcification depends on the presence of complication or degeneration. Computed tomography (CT) generally depicts a soft tissue mass with variable enhancement, which distorts the uterine contour and may exhibit coarse peripheral or central calcification.2Typical magnetic resonance imaging (MRI) features of non-degenerated leiomyomas include a well-circumscribed mass of homogeneously low to intermediate signal intensity (as compared to myometrium) on T1-weighted images, decreased signal intensity on T2-weighted images with surrounding flow voids and variable contrast enhancement. Degenerated leiomyomas have variable but characteristic appearances depending on the type of degeneration. Defining features include high T1 signal intensity or an irregular, T1-hyperintense rim around a centrally located leiomyoma in red degeneration; high T2 signal intensity in cystic degeneration; low T2 signal intensity with poor enhancement in hyaline degeneration; and very high T2 signal intensity with gradual enhancement on T1-contrasted sequences in myxoid degeneration.7 but they are rarely reported in pregnancy.In this case, a subserosal leiomyoma was misdiagnosed as an ovarian tumour antenatally. There are multiple reported cases of ovarian tumour-like leiomyomasA 34-year-old gravid female was referred to a tertiary hospital at 38+ weeks with a large cystic pelvi-abdominal (PA) mass detected on ultrasound. She had no known medical co-morbidities and one previous uneventful pregnancy culminating in a normal vaginal delivery (NVD) at full term. On presentation, the patient was asymptomatic and examination revealed only an increased symphysis-pubis fundal height (SFH) for expected gestation. Urine analysis showed proteinuria and blood tests revealed a minimally elevated cancer antigen-125 of 48 kU/L  and a normal carcinoembryonic antigen level of 0.7 ng/mL .Transabdominal ultrasound showed a thick-walled cystic mass measuring approximately 25 cm × 14 cm × 15 cm with no internal vascularity. The mass was assumed to be ovarian in origin. A singleton intra-uterine pregnancy with foetal heartbeat was confirmed. A transvaginal ultrasound scan was not performed.The decision was made to induce labour based on the unclassified proteinuria and the clinical suspicion of possible underlying pre-eclampsia. During the induction of labour, foetal monitoring showed subtle signs of foetal distress, which warranted continuous monitoring, but despite in-patient care and continuous foetal monitoring, there was subsequent intra-uterine foetal demise and the patient delivered a stillborn baby via NVD. Following delivery, the patient’s condition deteriorated in the ward, developing a persistent unexplained tachycardia of approximately 150 bpm, a blood pressure of 156/119 mmHg and a serum calcium level well above normal limits . Emergent cross-sectional imaging in the form of contrasted CT abdomen showed a large cystic mass with a thick, irregular enhancing wall and septations , which wExploratory laparotomy revealed a large, friable, thick-walled, pedunculated fundal mass, left ovarian vein thrombosis, necrosis of the left ovary and broad ligament and offensive intra-uterine contents. The decision to proceed to total abdominal hysterectomy and left salpingo-oophorectomy was taken based on significant intra-operative bleeding, friable necrotic tissue and haemodynamic instability. Histopathological evaluation revealed a subserosal uterine leiomyoma with extensive hyaline degeneration and necrosis, left ovarian vein thrombosis, haemorrhagic infarction of the left ovary and endometrial and myometrial necrosis.No patient identifiable information has been presented. Informed verbal consent was obtained from the patient prior to publication.2 Although usually asymptomatic, some pedunculated subserosal leiomyomas may undergo torsion, resulting in infarction. Rare but recognised complications linked to uterine leiomyomas in pregnancy include an increased risk of spontaneous abortion, foetal malpresentation, placenta previa, preterm birth, caesarean section and peripartum haemorrhage.6 Placental abruption is a rare but potentially devastating outcome that has been inconsistently associated with uterine leiomyomas.6 The strongest correlation with placental abruption has been documented in patients with submucosal or retroplacental leiomyomas.5Leiomyomas are characterised by location, whether submucosal, intramural (within the myometrium) or subserosal. The latter are a subtype that may become pedunculated, projecting out of the uterine contour and mimicking an adnexal mass.Ultrasound is employed as the primary diagnostic imaging modality because of its accessibility, safety and bedside application. Ultrasound gives information on leiomyoma size, characteristics and complications. Cross-sectional imaging is usually not required for initial diagnosis in typical cases and is reserved for patients with diagnostic uncertainty or suspected complications.8 Difficulties in detection can occur with smaller tumours, those that extend into the uterine cavity, those that occur only in the lower segment and in the absence of intra-lesional calcification. Internal heterogeneity depends on the type and degree of degeneration. A cystic appearance occurs with various forms of degeneration and is generally attributed to tumour growth beyond its vascular supply.9 Because of the varied imaging patterns, degenerating leiomyomas often pose a diagnostic challenge, as in the case of our patient. Hyaline degeneration is the most common type of degeneration, accounting for 60% of cases. Cystic degeneration is observed in 4% of leiomyomas and is the terminal sequelae of oedema.10Coarse intra-lesional calcification, although an uncommon finding, remains the most specific sign for leiomyomas.11Confirming the diagnosis of ovarian vein thrombosis with ultrasound is highly operator dependent and may be confounded by patient factors such as overlying bowel gas and poor patient cooperation. Contrasted abdominal CT is sensitive and specific for ovarian vein thrombosis and should be considered as the initial investigation of choice in the absence of pregnancy. Computed tomography features include visualisation of a tubular retroperitoneal mass with central low attenuation extending cephalic to the inferior vena cava or left renal vein.14 The presumed pathophysiology is leiomyoma-induced elevated serum parathyroid hormone-releasing protein (PTHrP), with a likely causal relationship proven in all but one case.13 In our patient’s case, serum PTHrP was not tested and the role of hypercalcaemia in the eventual outcome cannot be accurately extrapolated because of many concurrent variables. However, it should certainly encourage further research into PTHrP-secreting leiomyomas.Leiomyoma-associated hypercalcaemia is a rarely described entity, with seven reported cases to date.2This case highlights the importance of employing multiple imaging modalities when confronted with a PA mass of uncertain origin in a pregnant patient. It also underscores the importance of a thorough search for the ovaries when imaging a PA mass, although this search may be hampered by distortion and displacement by large masses. Magnetic resonance imaging is the most accurate modality to evaluate tumour characteristics and may be utilised in the pregnant patient when ultrasonography is inconclusive.Leiomyomas in pregnancy are common and are usually easily diagnosed, with no expected adverse obstetric complications. However, in rare cases, they may present in unpredictable ways with variable imaging findings that delay diagnosis and result in potentially harmful outcomes. Pedunculated uterine leiomyomas should be considered in the differential diagnosis of any PA mass. As degenerating leiomyomas are great mimics of other gynaecological and non-gynaecological medical conditions, familiarity with the different types of degeneration and their sonographic appearance is imperative to aid accurate diagnosis.Ultrasound should be employed as the initial imaging modality of choice for PA masses in pregnancy. However, its limitations should be carefully considered and cross-sectional imaging should be used where appropriate.


Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors. Fluorescent proteins (FPs) form the backbone of modern intracellular biosensors. Genetic encoding permits utilization of the natural protein expression machinery to allow cell-type specific labeling, even in whole organisms. Fusion to biosensing protein domains also enables the reporting of dynamic subcellular events, such as changes in second messenger concentrations or post-translational modifications. Such biosensors are designed to modulate either the intensity of a single fluorescent protein or energy transfer between two different fluorophores. Each of these design strategies has different strengths. Intensity-modulated sensors are optimal for reporting the patterns of rapidly changing phenomena, such as action potential-driven calcium changes in neurons . RatiomeThe two fluorescent protein design-strategy relies on transferring energy from the excited state of the donor fluorescent protein to the unexcited acceptor fluorescent protein, which is then free to emit a photon. Energy migration proceeds through the Förster resonance energy transfer (FRET) mechanism. Importantly, FRET efficiency is highly sensitive to both the separation distance and the relative orientation of the FRET pair ,9,10. DiDifferently colored donor and acceptor fluorophores are typically incorporated into FRET-based biosensors ,14. SuchBoth of these issues can be resolved by re-engineering two-color sensors to incorporate fluorescent proteins of the same color. FRET between two identical fluorescent species, known as homotransfer, was first observed in the early 20th century ,20, wellCurrent methods of homotransfer FRET measurement use polarized light to separate photons from the donor and acceptor fluorophores ,24. PolaThe fluorescence illumination must first be linearly polarized to measure homotransfer FRET using fluorescence polarization microscopy. Light waves have oscillating electric and magnetic fields that arise perpendicularly to each other. The extent that an electric field oscillates with spatial uniformity is its polarization. The modern convention is to describe planar polarization relative to the orientation of the light’s electric field. Linear polarizers that restrict the electric fields to a single direction a are theThe absorption of light by organic fluorophores is selective for specific light polarizations . Since lThe extent that emission retains its polarization during photoselection is a function of how quickly the dye can rotate during the lifetime of the excited state. Fluorescence lifetimes generally last a few nanoseconds. Small fluorophores in aqueous solutions, such as fluorescein, have rotational diffusion times in the hundreds of picoseconds. Extensive movement during the excitation state produces essentially isotropic fluorescence , indicated by a loss of measurable polarization. Large fluorescent proteins, on the other hand, rotate much more slowly and have rotational diffusion times that are roughly 10-fold greater than their fluorescence lifetimes ,32,33. CThe constraint placed by molecular size on fluorescent protein anisotropy is so strong that the only practical method to depolarize fluorescence is to transfer energy to a second molecule outside the photoselection plane through FRET c. FurtheThe “efficiency” of FRET between two fluorophores is the fraction of donor molecules that transfer energy to acceptors. There are several available methods for calculating two-color FRET efficiency, including fluorescence lifetime measurements of the donor ,36, photP) and perpendicular (S) intensity, over the total fluorescence:P and S intensities:Homotransfer FRET, however, can be quantified by measuring fluorescence polarization, since FRET efficiency is proportional to the depolarization of fluorescence emission. Polarizations are conventionally described by their anisotropy  and perpendicular (S) components may be simultaneously collected with a single camera using an image-splitting device. Polarization-compatible devices are available from Optical Insights , Hamamatsu (W-view Gemini), and Cairn (Opto-split), and we have implemented each of these solutions successfully. Alternatively, P and S images can be collected sequentially using a filter wheel to exchange suitably oriented polarizers.After the emitted light passes through the fluorescence filter cube, images containing parallel  , which i embryos . Image cThe most straightforward method for constructing a homotransfer reporter is to convert a validated heterotransfer biosensor by swapping out the fluorescent proteins for a homotransfer pair. Calcium biosensors were among the first converted to homotransfer ,55, but Several biosensors of varied design have been successfully converted to homotransfer reporters, demonstrating the robustness of the approach. Classic “molecular switch” type sensors, such as the Protein Kinase A activity reporters (AKARs) that contain both external sensing domains and internal effector-binding domains, have been converted to homotransfer reporters in four different colors . SensorsHomotransfer reporters can be optimized using the same strategies developed for heterotransfer reporters. One approach that is particularly noteworthy is the use of circularly permuted fluorescent proteins. Such variants are generated by fusing the natural N and C termini of the fluorescent protein, and creating alternate starting and ending positions in the fluorescent protein sequence . IncorpoCircularly permuted versions of the mCerulean3 CFP  and the Biosensors can also be created using a single fluorescent protein label  since bi2+/calmodulin-dependent protein kinase II has been observed during its activation using homotransfer [Conformational changes within protein complexes have also been resolved using homotransfer. Structural rearrangement of singly-labeled Catransfer . Homotratransfer .Single-color sensors can also be engineered using rational design. The Apollo nicotinamide adenine dinucleotide phosphate (NADP+) probe uses a catalytically inactive glucose-6-phosphate dehydrogenase mutant as the sensing domain . DimerizOne of the principal advantages of employing homotransfer reporters is the ability to accommodate secondary fluorescence and optogenetics tools. It remains challenging to measure multiple two-color FRET sensors in the same cell at the same time. FRET reporters generally use either the cyan/yellow or green/red FP combinations. Since these pairs take up a broad swath of the useful visible light spectrum, pairing them with a second FRET reporter may require stretching into the near-infrared . FurtherHomotransfer sensors, however, can be more easily switched to optically compatible areas of the spectrum. For example, the Apollo NADP+ sensor was converted from YFP to CFP for use with a green peroxide indicator . AdditioIntravital, multiplexed imaging of FLAREs has also been performed using two-photon microscopy . The bioThe spectral flexibility of homotransfer reporters is also advantageous for combination with optical reporters. Light-gated ion channels, such as the ChRs, tend to have absorbances that overlap with CFP, GFP, and YFP. For example, a commonly used ChR has a peak excitation at approximately 460 nm , making The flexibility of the homotransfer sensors also permits them to be used with other optogenetic tools such the photo-inducible protein assembly using the light-oxygen-voltage-sensing (LOV) domain . Fusion Despite less overlap between the donor and emission spectra, homotransfer reporters still compare favorably to heterotransfer biosensors in generating contrast. The signal-to-noise ratios of the sensors, as measured by the maximal change in response divided by the standard deviation of the basal state  ranges fThe nature of the data collected for polarized FRET is also advantageous for quantitative analysis because channel crosstalk is eliminated. For heterotransfer FRET, acceptor excitation  and donoWe first used automated image analysis of homotransfer data to study protein-protein interactions in the endoplasmic reticulum (ER). The sarco(endo)plasmic calcium ATPase (SERCA) pumps calcium into the ER/sarcoplasmic reticulum lumen, and it is regulated by several small transmembrane peptides , includiTo examine the effect of SERCA interaction on SLN–sANK1:mVenus homotransfer across many cells, we developed a computationally automated analysis approach . PreviouWe used the automated pixel-by-pixel analysis in combination with a two-fluorescent protein biosensor for glucokinase activity , which uC. elegans [We performed a similar analysis to track myosin phosphorylation in developing  elegans . Here, wLarge-scale pixel level analyses are much more difficult to apply to heterotransfer FRET data, at least in our experience. Although numerous corrective methodologies ,79,80 haP and S, Fluorescence polarization microscopy also provides an alternative for FRET image representation . TypicalS channel is isotropic by definition, it can be subtracted from the P image to highlight regions with the most polarization. Further, the S channel can be multiplied by a normalization factor to mask areas of less than the desired anisotropy threshold [Intensity information can be preserved in polarization FRET images using image subtraction, rather than image ratioing. Since the hreshold . NormaliHomotransfer reporters offer significant advantages over heterotransfer biosensors, including increased measurement precision, flexibility for multiplexing, and improved methods for data analysis. Furthermore, a large number of validated heterotransfer biosensors have been converted to homotransfer reporters, and they are readily available for biological experimentation. Recent advances in data analysis and optical sectioning have also expanded the power of the FRET-based biosensor approach and can now be used to explore biological questions in complex physiologic systems.


Endoscopic ultrasound (EUS)-guided treatment has been recently described for internalizing refractory pancreaticocutaneous fistulas (PCFs). However, the existing techniques are limited because of the difficulty in accessing nondilated pancreatic ducts or fistulas. In an attempt to overcome this limitation, we present a case where a EUS-guided intervention utilizing a balloon-target technique was employed to internalize a PCF into the stomach.A 78-year-old woman underwent percutaneous drainage and 4 percutaneous endoscopic necrosectomies for walled-off pancreatic necrosis (WOPN) after severe acute pancreatitis due to choledocholithiasis. Although the WOPN was resolved, refractory PCFs remained.Pancreaticocutaneous fistulas.An echoendoscope was introduced into the stomach, but the narrow PCF lumen made visualization of the fistula by EUS difficult. Subsequently, a balloon catheter was percutaneously inserted into the fistula, and then the inflated balloon was visualized by EUS from the stomach. The balloon was punctured with a 19-gauge fine needle through the posterior wall of the upper body of the stomach . A guidewire was then passed through the fistula to the outside of the body through the EUS scope. After dilating the gastro-fistula space with an 8-mm balloon dilation catheter, a 7-French double pigtail catheter was placed from the stomach into the PCF.The percutaneous drainage tube was removed after one week, and the patient was discharged 6 months after admission. No adverse outcomes have been observed in the 2 years since the procedure.PCFs can be successfully managed using EUS-guided internalization with a balloon-target technique. Severe necrotizing pancreatitis and WOPN can lead to complete duct disruption and subsequent main pancreatic duct disconnection.,3 When percutaneous drainage and necrosectomy are performed for WOPN management, pancreaticocutaneous fistulas (PCFs) can develop from the upstream pancreatic duct through the drain site after the drainage has been completed. The standard endoscopic method for evaluating and treating PCFs is endoscopic retrograde cholangiopancreatography (ERCP).,5, but this may fail due to impassable ductal strictures or the inability to effectively bridge a pancreatic duct disruption with a stent. Recently, endoscopic ultrasound (EUS)-guided treatments have been described for internalizing refractory PCFs.,7 However, the existing techniques are limited due to difficulty in accessing non-dilated pancreatic ducts or fistulas. Here, we present a case where this limitation was overcome by employing an EUS-guided intervention with a balloon-target technique, providing an alternative drainage pathway that allowed the cutaneous fistula to heal.Acute necrotizing pancreatitis has been reported in approximately 20% of pancreatitis episodes and has a mortality rate of 10% to 30%. Walled-off pancreatic necrosis (WOPN) occurs in 1%–9% of acute pancreatitis cases, usually 4 to 6 weeks after the initial episode. Without procedural intervention,2 Treatment with continuous intravenous infusion of nafamostat mesilate (240 mg/day), intravenous infusion of meropenem (2.0 g/day), and intravenous infusion of approximately 5000 mL/day was initiated. Two days later, a re-examination CT scan showed a lack of arterial enhancement from the pancreatic body to tail, and pancreatic arterial infusion therapy was administered for 1 week. Four weeks after admission, the patient developed pyrexia of approximately 40°C, and WOPN was suspected on CT scan. Accordingly, percutaneous drainage was performed from the left intercostal space, and a 12-French pigtail drainage catheter  was placed. However, drainage of the necrotic material was insufficient, and a percutaneous endoscopic necrosectomy was performed for WOPN 3 months after admission. The WOPN was resolved after 4 necrosectomies, and endoscopic extraction of the common bile duct stones was performed. Unfortunately, the PCF remained  and serum trypsin levels of 8465 ng/mL (reference range 100–550 ng/mL), suggested acute pancreatitis. An increased white blood cell count of 23,900/μL (reference range 3300–8600/μL), indicated severe inflammation. Contrast-enhanced CT revealed pancreatomegaly, effusion extending from the peripancreatic space to the pelvic cavity, and calcified stones in the lower portion of the common bile duct. The patient was diagnosed with acute pancreatitis due to gallstones and was admitted for treatment. An APACHE II score of 12 led to the diagnosis of severe acute pancreatitis.3 External pancreatic fistulas, known as PCFs, generally result from the placement of a percutaneous drain to collect pancreatic fluid, as occurs in WOPN treatment. The fistula is likely to persist if there is complete transection of the pancreatic duct, a downstream ductal stricture, or if it is a high-output fistula (>500 mL/day). Nonsurgical therapies for pancreatic fistulas include bowel rest, total parenteral nutrition, jejunal feeding, and somatostatin analogs such as octreotide. However, there is limited evidence to support these interventions.,10Severe necrotizing pancreatitis and WOPN can result in complete duct disruption, causing disconnected pancreatic duct syndrome with a viable upstream pancreas segment draining out of a low-pressure fistula created surgically or by a percutaneous catheter.,5 However, ERCP may fail in patients with disconnected pancreatic duct syndrome where a segment of the pancreatic duct in the tail is completely disconnected from the downstream duct in the pancreatic head. The management options for these patients are long-term percutaneous drain placement or surgery, both associated with substantial morbidity. The access to pancreatic ducts or fistulas via EUS guidance enables the treatment of PCFs that are not amenable to ERCP.,7 This EUS-guided rendezvous technique is carried out by the puncture of an EUS needle into the pancreatic duct followed by the passage of a guidewire via the needle and across the ductal obstruction to the gut lumen, facilitating pancreatic stent placement via ERCP. Similarly, EUS-guided transmural stent placement involves positioning a pancreatic guidewire via an EUS needle. The area from the transgastric or transduodenal tract to the pancreatic ducts or fistulas is then dilated, and a stent is placed via the tract. Irani et al pioneered a combined EUS and interventional radiologic technique for the placement of internal stents in PCF patients, in which the fistula tract rather than the pancreatic duct is drained, and much of the manipulation is performed percutaneously with endoscopic or EUS guidance. If the pancreatic duct is dilated or the pancreatic fistula is large, an EUS-guided puncture from stomach or duodenum is easier. However, the pancreatic duct is frequently decompressed by the fistula and usually narrow, preventing access for fluid collection. This paper presents a transgastric balloon-target technique for accessing narrow PCFs using EUS, allowing for placement of a drain from the PCF to the stomach. If the PCF is narrow, a balloon can be placed into the fistula percutaneously, providing a target for EUS-guided puncture and fistula drainage. The balloon placed in the fistula can be clearly visualized by EUS, allowing an easier and safer EUS-guided puncture. One technical challenge generally encountered is difficulty in performing EUS-guided puncture when the blood vessel or intestinal tract is positioned between a fistula and the stomach.ERCP is the primary endoscopic modality for PCF treatment, and the placement of a pancreatic duct stent that bridges the ductal disruption often successfully resolves the fistula.4In conclusion, PCF can be successfully managed using EUS-guided internalization with a balloon-target technique. Creating an alternative drainage pathway from the pancreatic fistula into the stomach diverts pancreatic fluid away from the PCF, allowing healing and obviating the need for long-term percutaneous drain placement or surgery.We thank the staff of the endoscopy at the Kashiwazaki General Hospital and Medical Center for cooperating in the treatment.Conceptualization: Michitaka Imai.Formal analysis: Michitaka Imai.Investigation: Michitaka Imai, Yoshifumi Takahashi.Methodology: Michitaka Imai, Toshihiro Sato.Supervision: Michitaka Imai.Validation: Michitaka Imai, Masaki Maruyama.Writing – original draft: Michitaka Imai.Writing – review & editing: Osamu Isokawa.


Clinical tuberculosis diagnosis and empiric treatment have traditionally been common among patients with negative bacteriologic test results. Increasing availability of rapid molecular diagnostic tests, including Xpert MTB/RIF and the new Xpert Ultra cartridge, may alter the role of empiric treatment.We prospectively enrolled outpatients age > = 15 who were evaluated for pulmonary tuberculosis at three health facilities in Kampala, Uganda. Using sputum mycobacterial culture, interviews, and clinical record abstraction, we estimated the accuracy of clinical diagnosis relative to Xpert and sputum culture and assessed the contribution of clinical diagnosis to case detection.Over a period of 9 months, 99 patients were diagnosed with pulmonary tuberculosis and subsequently completed sputum culture; they were matched to 196 patients receiving negative tuberculosis evaluations in the same facilities. Xpert was included in the evaluation of 291 (99%) patients. Compared to culture, Xpert had a sensitivity of 92%  and specificity of 95% (92–98%). Twenty patients with negative Xpert were clinically diagnosed with tuberculosis and subsequently had their culture status determined; two (10%) were culture-positive. Considering all treated patients regardless of Xpert and culture data completeness, and considering treatment initiations before a positive Xpert (N = 4) to be empiric, 26/101 (26%) tuberculosis treatment courses were started empirically. Compared to sputum smear- or Xpert-positive patients with positive cultures, empirically-treated, Xpert-negative patients with negative cultures had higher prevalence of HIV (67% versus 37%), shorter duration of cough (median 4 versus 8 weeks), and lower inflammatory markers (median CRP 7 versus 101 mg/L).Judged against sputum culture in a routine care setting of high HIV prevalence, the accuracy of Xpert was high. Clinical judgment identified a small number of additional culture-positive cases, but with poor specificity. Although clinicians should continue to prescribe tuberculosis treatment for Xpert-negative patients whose clinical presentations strongly suggest pulmonary tuberculosis, they should also carefully consider alternative diagnoses. Empiric treatment for tuberculosis (TB), in the absence of bacteriologic confirmation, has long been a common practice made necessary by the limited sensitivity of available diagnostic assays ,2. ConveXpert has led to more bacteriologic confirmation among patients treated for TB , but it Mycobacterium tuberculosis.We evaluated the role of clinical diagnosis (i.e. diagnosis without bacteriologic confirmation) among adults being evaluated for pulmonary TB in a real-world high HIV- and TB-burden setting where on-site use of Xpert is well established. We observed the results of Xpert and other diagnostic testing and incorporation of these results into clinicians’ treatment decisions. We subsequently performed sputum culture (as a gold standard), and measured the sensitivity and specificity of Xpert and of the overall clinician treatment decision for culture-positive TB. We report on the additional contribution of clinical diagnoses to TB case detection and on the characteristics of empirically-treated patients whose sputum cultures were ultimately negative for Participants were enrolled from three health facilities  that had been identified as the primary clinics providing TB care to residents of neighboring parishes in Kampala, Uganda. The two largest facilities had on-site GeneXpert devices and were preparing to transition to the use of Xpert Ultra cartridges, and the third referred its specimens to nearby clinical or reference laboratories for Xpert.Between May 2018 and February 2019, we recruited patients aged ≥15 years who were diagnosed with pulmonary TB at these health facilities; eligibility for TB culture was restricted to residents of three parishes nearest the health facilities, except during a six-month period at one health facility when no residence-based inclusion criteria were imposed (in order to expand sample size). For the current analysis, we excluded patients for whom TB treatment was initiated based on a history of partial TB treatment and previous loss to follow-up. For each enrolled case, we randomly selected two controls aged ≥15 years, matched by health facility and residence , from among those patients who were evaluated for pulmonary TB and determined not to have TB on a selected day following the case’s enrollment. Diagnosis and treatment decisions were made by routine clinical staff, and eligible individuals were subsequently identified by research study staff via review of the health facilities’ presumptive TB registers after decisions to treat or not treat for TB had been made. Health facility clinicians retrospectively confirmed that all enrolled cases had been diagnosed with pulmonary TB and recommended for treatment, and that all controls had been evaluated for suspected pulmonary TB but neither diagnosed with, nor treated for, TB at the end of that evaluation.Patients were invited to enroll in the study on the day that they were informed of the clinical decision for or against a TB diagnosis. After providing written informed consent , enrolled cases and controls participated in a standardized interview that included TB symptoms, risk factors, and care seeking. They provided expectorated sputum for mycobacterial culture (described below) and venous blood for CRP measurement  at the time of study enrollment. Neither test was part of standard care; the tests were performed after clinician decisions to treat or not treat for TB, and clinicians were not informed of culture or CRP results except in instances when an enrolled control (a patient not treated for TB) had a positive sputum culture result. Medical records were abstracted to collect information about the TB evaluation including any Xpert or other diagnostic testing, any HIV and ART history, and results of HIV testing which was routinely performed as part of TB diagnostic evaluation.N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH) digestion and decontamination  cultures were positive), 8 received 2–7 days of treatment before culture (2 [25%] cultures positive), and 2 received 8–14 days of treatment before culture (2 [100%] cultures positive).Culture status could be determined for 293 (97%) study participants: 99 cases (including 96 cases with Xpert results) and 1894 controls Unavailable culture results included one case who could not produce additional sputum for culture (but who had a positive sputum Xpert result)and one case and six controls  for whom both solid and liquid cultures were contaminated. Liquid culture contamination was common (13%), but solid culture contamination was rare (3%), and there was otherwise good agreement between liquid and solid culture .Of the 289 patients with a definitive sputum culture result , sputum culture was positive in 71 (72%) of 99 patients enrolled as cases and 4 (2%) of 181 enrolled as controls.Clinicians’ diagnostic decisions (prior to availability of a culture result) and patients’ subsequent culture results are illustrated for all empirically-treated patients with a definitive culture result in 26 (26%) treated patients were treated empirically : 10 wereIn sensitivity analysis, there was minimal change in the estimated sensitivity and specificity of Xpert if all patients with contaminated or missing cultures (N = 7) were assumed to have culture result that corresponded to their Xpert results  or to thCharacteristics of patients with discrepant Xpert and culture results are shown in Finally, empirically treated, culture-negative cases were more likely than other treated patients to have started treatment before they provided sputum to the research study for culture , but this treatment before culture was similarly common among culture-positive empirically treated patients  and culture-negative empirically treated patients  and may reflect the perceived urgency of treatment for those empirically treated.The increasing worldwide use of Xpert and the ongoing transition to Xpert Ultra present the potential to reduce reliance on clinical diagnosis and to reduce empiric treatment for patients with negative bacteriologic results. The appropriate clinical response to the availability of on-site molecular TB testing depends on the performance of Xpert in a given patient population and clinicians’ ability to identify the remaining patients with TB who are missed by Xpert. In our evaluation of 300 presumptive pulmonary TB patients in an outpatient setting of high TB and HIV burden, we observed a high sensitivity and specificity for Xpert relative to sputum culture, such that very few  culture-confirmed TB cases were missed by Xpert. Clinical diagnosis and empiric treatment after negative Xpert identified two additional culture-confirmed TB cases, but at the expense of prescribing treatment to 18 patients with negative cultures for TB.These results show that Xpert can perform as a useful diagnostic test in routine clinical care in a high-burden, low-resource setting, even before transition to the Ultra cartridge. Xpert provided bacteriologic diagnosis of over 90% of patients presenting with sputum-culture-positive TB, including over 80% of those with HIV coinfection. Ultra is expected to increase this sensitivity further, particularly for patients with HIV, although it may also reduce somewhat the high specificity that we observed . In advoWith Xpert performing accurately in practice and becoming increasingly available at the point of care in high-burden settings such as Uganda, it may be appropriate to reevaluate empiric treatment practices. Country-wide, Uganda’s non-bacteriologic TB diagnoses remained stable between 30 and 35% from 2013 to 2017 despite expanding Xpert use , consistIt is admittedly challenging to define how much empiric overtreatment is too much, and at what point the risks and costs of possible overtreatment outweigh the benefits of empiric treatment for a few patients. The 10% culture-positive rate that we observed among clinically-diagnosis patients is still four-fold higher than the probability of selecting a culture-positive individual by chance from our eligible Xpert-negative care-seeking study population (estimated at 2.5% based on a register review that suggested 6.0 eligible controls per enrolled case). Clinicians, even knowing that TB is unlikely in a given patient, may hesitate to defer treatment in patients with risk factors for high mortality or poor follow-up, or when they lack a treatable alternative diagnosis. It is also true that sputum TB diagnostics including culture are imperfect, that some of the patients with negative sputum cultures in our study may have had culture-negative TB disease that benefitted from empiric treatment, and that the judgment of experienced clinicians is a valuable tool for recognizing high-risk, sputum negative TB presentations. Notably, however, we found that most clinically-diagnosed, culture-negative patients were less sick than the typical TB case based on symptoms and inflammatory markers, suggesting treatment could have been deferred in these patients  with little expected harm. Jumping quickly to empiric TB treatment risks missing important non-TB diagnoses. In addition, in advanced HIV where clinicians may feel most compelled to treat empirically, emerging evidence shows a limited role for empiric TB treatment in reducing mortality .Our study has some important limitations. Due to limited detail of clinical record-keeping and limited accessibility of patient records beyond the on-site diagnostic and treatment registers, we were unable to fully characterize the reasoning behind empiric treatment decisions. More extensive collection and expert review of data about empirically-treated, culture-negative patients–including the clinical evidence behind their diagnoses, the extent of their response to TB treatment, and the existence of alternative diagnoses–could help to elucidate the probability that such patients had true, culture-negative TB disease. In addition, although we have used sputum culture as a gold standard for diagnosis of pulmonary TB, we recognize that culture is imperfectly sensitive. Potential reasons for falsely negative sputum cultures include extra-pulmonary TB , errors of laboratory processing, or antimicrobial treatment prior to culture. In our study, nearly one third of clinically diagnosed, culture-negative patients initiated treatment before sputum could be collected for culture, although most received no more than 3 days, which would be expected to reduce sputum CFU by less than one log  and to rIn summary, judged against sputum culture, we observed high sensitivity and specificity of Xpert in routine outpatient care in urban Uganda, confirming its reliability as the primary test for TB diagnostic evaluation in similar settings of high TB and HIV prevalence. Empiric treatment remained common, at levels potentially comparable to the pre-Xpert era. Clinical diagnosis allowed a small number of additional culture-positive cases to be treated, but it had poor specificity in identifying such patients. As clinics increasingly use Xpert for TB diagnosis and transition to the Ultra cartridge, clinicians may need to adjust their threshold for empiric treatment, by continuing to treat Xpert-negative patients whose clinical presentations are most suggestive of TB, but by also more strongly pursuing possible alternative diagnoses in most Xpert-negative patients.S1 FigCRP measurements of clinically-diagnosed, culture-negative patients , compared to smear/Xpert-positive culture-positive cases  and to smear/Xpert-negative culture-negative cases . Lower and upper bounds of the assay used are 2.5 and 300 mg/L.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Cucurbita pepo pathosystem indicates that infection with Zucchini yellow mosaic virus (ZYMV) reduces exposure to a second insect-vectored parasite (Erwinia tracheiphila). In this study, we performed two large-scale field experiments employing wild gourds (Cucurbita pepo ssp. texana), including plants with a highly introgressed transgene conferring resistance to ZYMV, to examine the interaction of ZYMV and powdery mildew, a common fungal disease. We found that ZYMV-infected plants are more resistant to powdery mildew . As a consequence, during widespread viral epidemics, proportionally more transgenic plants get powdery mildew than non-transgenic plants, potentially mitigating the benefits of the transgene. A greenhouse study using ZYMV-inoculated and non-inoculated controls (non-transgenic plants) revealed that ZYMV-infected plants were more resistant to powdery mildew than controls, suggesting that the transgene itself had no direct effect on the powdery mildew resistance in our field study. Additionally, we found evidence of elevated levels of salicylic acid, a phytohormone that mediates anti-pathogen defenses, in ZYMV-infected plants, suggesting that viral infection induces a plant immune response (systemic acquired resistance), thereby reducing plant susceptibility to powdery mildew infection.Few studies have examined the combined effect of multiple parasites on host fitness. Previous work in the  Many studies have examined the costs of parasite infection for host fitness. In both plant and animal systems, parasites can increase mortality, reduce reproductive output, reduce growth, and induce defense systems that are costly in terms of energy and nutrients e.g., ,b. The pDirect interactions, such as interference competition between parasites, have been well documented . HoweverPre-infection interactions among competing parasites have been documented in a few study systems. “Ecological interference” has been proposed as a mechanism of interaction in directly transmitted childhood infections, whereby illness due to a primary infection reduces contact rates and therefore exposure to a secondary infection , 2003. VAfter infection, a co-infecting parasite enters an environment in which host resources may already be consumed by an initial parasite, or in which host defenses are upregulated in response to the primary infection. In plant cells, infection can trigger signaling cascades of the innate immune system that provide broad spectrum immunity against subsequent parasite attack . These sCucurbita pepo ssp. texana  is an annual monoecious vine that is cross compatible with cultivated squash and several annual species of wild Cucurbita , a wind-dispersed fungal pathogen of squash, cucumbers and melons. The fungus overwinters as chasmothecia  which serve as the source of initial infection each year. However, conidia  are the primary means of dispersal. Symptoms first appear on older leaves as small reddish-brown spots that quickly turn white as hyphae are produced. As the disease progresses, leaves become covered in the powdery, white mycelia before shriveling and dying. In central Pennsylvania, first symptoms typically occur in early August and by September the disease will spread to the entire population.At our field sites in Central Pennsylvania, the Erwinia tracheiphila, i.e., a pre-infection interaction, by changing the foraging behavior of the beetle vector  into 20 families of the wild gourd (using the wild gourd as the recurrent parent) because the presence of the NPTII protein in hybrid progeny can be detected serologically. Previous studies have shown that the introgressed transgene effectively deters the target viruses under field conditions  is hemizygous and, importantly, the nditions , 2010a. texana) plants, 9 tBC9, and 9 ntBC9 from each of five families  into each of four 0.4 ha fields at the Pennsylvania State University Agriculture Experiment Station at Rock Springs, PA. In two of the four fields (located 25 m apart) we inoculated eight wild gourd and four ntBC9 from each of the five families prior to transplanting. Inoculum was prepared by grinding 2–3 ZYMV infected symptomatic leaves in 100 ml of a phosphate buffer. Plants were then inoculated by brushing the adaxial leaf surface with 320 grit carborundum and then dipping a small pestle into the inoculum and gently rubbing the leaf surface in a circular motion. This inoculation design resulted in 60 inoculated plants, 75 non-inoculated wild gourds and ntBC9 (virus susceptible) plants, and 45 tBC9 plants per field . Symptoms first appeared 7–10 days after inoculation. The other two fields had no inoculated plants and were located approximately 1 km from the fields with the inoculated plants. The non-inoculated fields were also separated by 25 m and they were permitted to become naturally infected with mosaic viruses. In all four fields, the plants were the progeny from the same five randomly selected families . Moreover, the tBC9 and ntBC9 from each family were full siblings  as were all of the wild gourd plants from a given maternal parent. In all four fields, plants were allowed to become infected with powdery mildew naturally.We germinated seeds in a greenhouse in early May 2013 and 2014 and, in late May of each year, we transplanted 18 wild gourds  it was not possible to find 20 symptomatic plants in early July. In no case did our field diagnosis differ from the immunological test results. However, it should be noted that the ELISA based ImmunoStrips are not as sensitive as RT-PCR screening. Consequently, it possible that some asymptomatic plants had low titers of ZYMV (Podosphaera sp. (most likely Podosphaera xanthii). We assessed powdery mildew symptom severity on a 0–3 scale where 0 indicates a plant showing no powdery mildew symptoms, 1 indicates a plant with <33% of leaves showing >50% spread of powdery mildew symptoms on the leaf surface, 2 indicates a plant with 33–67% of leaves showing >50% spread of powdery mildew symptoms on the leaf surface, and 3 indicates a plant with >67% leaves showing >50% spread of powdery mildew symptoms on the leaf surface. We assessed reproductive output by counting staminate  and pistillate  flowers weekly  until the beginning of September.The plants in each of the four fields were monitored once weekly throughout the growing season (beginning in early June) for symptoms of viral diseases. Our field diagnoses based upon visual symptoms were confirmed in both early July and early August by taking leaf samples from up to 20 symptomatic and 20 asymptotic plants per field and testing the leaves using ZYMV ImmunoStrips of ZYMV  and wereTo confirm that the interaction between ZYMV and powdery mildew was not a direct result of transgene presence, we grew 320 wild gourd plants  in a greenhouse in September 2014. Additionally, the greenhouse environment allowed for a controlled environment where the only difference between plants prior to powdery mildew introduction was presence or absence of ZYMV. We inoculated 160 plants (16 plants from each of 10 families) with ZYMV at the second true leaf stage using the procedure described above . We mock-inoculated 80 (8 plants from each of 10 families) plants using just the inoculation buffer and left 80 plants as untreated controls (8 plants from each of 10 families). Plants were arranged in a randomized block design. Plants were treated with an insecticide to prevent leaf herbivory and ZYMV spread. Fourteen days after all inoculated plants first showed ZYMV symptoms, 12 additional plants with established powdery mildew infections were moved into the greenhouse and uniformly distributed. Fans were used to circulate the spores and ensure all plants were rapidly exposed to the powdery mildew inoculum. Plants were scored for powdery mildew symptoms every 2 days for 3 weeks.g. The dichloromethane layer, now containing the compounds of interest and the internal standards, was removed to a glass vial and dried under house air. Briefly, after solvent extractions of leaf tissue we derivatized SA to its methyl ester, which we isolated using vapor-phase extraction and analyzed by coupled gas chromatography-mass spectrometry with isobutane chemical ionization using selected-ion monitoring. We quantified amounts of total free SA using 100 ng of [2H6]SA   to use for phytohormone analysis. Leaf samples weighing 0.1 g were collected from these plants 10 days post-inoculation (prior to the introduction of powdery mildew) and were taken from new leaves that were systemically infected. Samples were immediately flash frozen in liquid nitrogen. After harvesting, the tissue samples received 100 ng of internal, isotopic standards and 400 μL of an aqueous, weakly acidic buffer solution while on ice. After grinding and mixing, each sample then received 1 mL of dichloromethane and was mixed and centrifuged for 2 min at 12,000 ×  Canada) .Our initial analyses revealed that the wild type gourds and the ntBC9 plants did not differ statistically in their incidence or severity of powdery mildew infection, nor in their flower production. Therefore, we combined these two plant types into a single “susceptible’ class for the analyses presented below. Plants that died before the first incidence of powdery mildew in our fields  were not included in these analyses.Figure 1A). In the non-inoculated fields, first ZYMV incidence occurred in late July. The transgene was effective against ZYMV infection, though a few transgenic plants did express mild symptoms ZYMV infection in August (Supplementary Table S1).In 2013, flowering began in late June and peak flower and fruit production occurred in August. Powdery mildew first appeared in mid-August and spread rapidly in all fields. Natural ZYMV spread in the inoculated fields began in mid-June, with peak spread occurring before first powdery mildew incidence  and the non-inoculated fields  in mid-August, with the same trend continuing in late August. We used a mixed-effects analysis of variance (ANOVA) to assess the effect of plant type (susceptible or transgenic), family (random), treatment (virus inoculated fields or no inoculation fields), and a plant type by treatment interaction on powdery mildew symptom severity. We found that transgenic plants not only had a higher incidence of powdery mildew infection, they also experienced significantly more severe powdery mildew symptoms than virus susceptible plants at each time point during the 2013 field season  . A mixed-effects model ANOVA showed there was not a significant plant-type by treatment interaction  for the final powdery mildew scoring date (late August). However, inoculation treatment was not a significant factor indicating that ZYMV spread in the non-inoculated fields had reached a point where there were no longer treatment (field inoculation) differences. Additionally, there were significant family differences for powdery mildew severity at both time points , with one family showing the most severe symptoms .In 2013, there were significant differences in the incidence of powdery mildew between the two plant types (susceptible and transgenic) in both the inoculated fields . The transgene was effective against ZYMV infection, though a few transgenic plants did express mild symptoms of virus in August.In 2014, flowering began in late June and peak flower production occurred in August. Powdery mildew first appeared in mid-August and spread rapidly in all fields. In the inoculated fields, natural ZYMV spread began in late June and peaked in late July, with most susceptible plants (wild gourd and ntBC9) infected before first incidence of powdery mildew. In the non-inoculated fields, first incidence of ZYMV was in mid-August, occurring simultaneously with powdery mildew infection and spreading rapidly  than on the susceptible plants. However, there was no significant difference in the incidence of powdery mildew on transgenic and susceptible plants in the non-inoculated fields  indicating that the transgene per se does not alter the resistance of plants to powdery mildew. However, by early September, after virus had spread through the non-inoculated fields, there were significant differences in the proportion of transgenic and susceptible plants that contracted powdery mildew .In mid-August 2014, the incidence of powdery mildew was significantly higher on transgenic plants in the virus inoculated fields  . The ANOVA also revealed a significant positive plant-type by treatment (inoculated vs. non-inoculated fields) interaction term indicating that transgenic plants have more severe powdery mildew symptoms in the ZYMV-inoculated fields . Additionally, there were significant family differences for powdery mildew severity , with one family showing the most severe symptoms .We found that transgenic plants not only had a higher incidence of powdery mildew infection, they also experienced significantly more severe powdery mildew symptoms than non-transgenic plants at each time point during the 2014 field season . Moreover, transgenic and susceptible plants that were not virus infected had a similar incidence of powdery mildew during the initial stages of the powdery mildew outbreak . We used a logistic regression model to assess the effect of plant type (transgenic or susceptible), family (random), treatment (inoculated or non-inoculated fields), and ‘weeks with virus symptoms,’ on the likelihood of infection with powdery mildew. This analysis showed that weeks with virus is a significant predictor of powdery mildew incidence and for each week a plant has been infected with virus, ZYMV-infected plants are 0.84 times less likely to contract powdery mildew. A reduced model that did not include weeks with virus showed that when powdery mildew first enters our fields, the odds of a transgenic plant contracting powdery mildew are 2.4 times that of susceptible plants (most of which are infected with ZYMV at this time in the season). However, the full model is a better fit  and ‘when weeks with virus’ is included in the model, plant type is no longer a significant predictor. This suggests that differences between transgenic and susceptible plants are a result of viral infection and not the transgene per se. The logistic regression model also revealed a significant family effect. In particular, one maternal family showed higher odds of contracting powdery mildew relative to the other four families suggesting that there was heritable (in the broad sense) variation for resistance to powdery mildew among families.Among susceptible plants in our fields, the proportion of plants that contracted powdery mildew during the initial outbreak decreased with the number of weeks they were infected with ZYMV (0–10 weeks) . We found that virus-infected plants showed significantly less severe powdery mildew symptoms than the other two treatments (mock-inoculated and control) and that mock-inoculated plants showed significantly more severe symptoms than the other two treatments (virus-infected and control) . There were significant family differences in powdery mildew severity as well  , indicating broad sense heritable variation for powdery mildew resistance among the 10 families used in this study. A significantly smaller proportion of virus-infected plants were infected with powdery mildew relative to the mock-inoculated and control treatments at the early  and middle  time points (Supplementary Table S2). However, at the conclusion of the experiment, all plants were infected with powdery mildew.The results of the 2013 and 2014 field experiments suggested that healthy (not virus infected) plants were more susceptible to powdery mildew and that the transgene had no direct effect on susceptibility to powdery mildew. To explicitly test these findings, we conducted a greenhouse experiment that consisted of three treatments  and used only wild gourds (no transgenic plants). A repeated measures ANOVA showed that treatment was a significant factor for predicting susceptibility to powdery mildew . A Tukey’s HSD test showed that leaves of ZYMV-infected plants contained significantly higher levels of SA compared to mock-inoculated and control plants .An ANOVA revealed significant differences in SA production among the treatments  in our greenhouse study . We did not conduct a regression analysis on the 2013 field data as there were only two powdery mildew scoring dates in 2013. However, an LS-means showed that susceptible plants produced 0.638 (SE = 0.012) of their total flower production and transgenic plants produced 0.628 (SE = 0.024) of their total flower production post first incidence of powdery mildew in our fields. We also used an analysis of covariance to assess the effects of weeks with powdery mildew (covariate), plant type (transgenic or susceptible), family (random), and treatment (inoculated or non-inoculated fields) on the proportion of both the total staminate and the total pistillate flowers produced by each plant after powdery mildew infection in both years. This analysis revealed that there was no difference in the proportion of flowers produced by transgenic and susceptible plants after powdery mildew infection in 2013 . In contrast, our findings revealed that in 2014, transgenic plants produced a greater proportion of both staminate and pistillate flowers after powdery mildew infection relative to susceptible plants . Moreover, even when weeks with virus is included as a second covariate in the model, transgenic plants still produce a significantly greater proportion of flowers than susceptible plants after powdery mildew infection . This suggests that although powdery mildew infection has a negative impact on fitness, due to the timing of the diseases in our fields, the impact of powdery mildew was not as great as the impact of virus infection on plant fitness.A regression analysis revealed that there is a slight (but significant) negative impact on plant fitness as a result of powdery mildew infection in 2014  plants infected with ZYMV are less likely to become infected with powdery mildew, (2) that when infected with powdery mildew, ZYMV-infected plants show reduced powdery mildew symptom severity, and (3) that infection with powdery mildew adversely affects the production of staminate and pistillate flowers. By August, when powdery mildew entered our ZYMV-inoculated fields, most non-virus-infected plants in our inoculated fields were transgenic individuals, however, our data show that the transgene has no direct effect on powdery-mildew infection . Additionally, the greenhouse experiment (which did not use any transgenic plants) also showed that virus infected plants are more resistant to powdery mildew than their non-infected siblings, further indicating that the interaction between ZYMV and powdery mildew is not a direct result of transgene presence Figure 4), similar to previous work . While we expected that mock-inoculated plants would have more severe symptoms than the virus-inoculated plants, the fact that they had more severe symptoms than the control plants was surprising. The leaf damage caused by the inoculation/mock-inoculation procedure (abrasion) may have provided an easier avenue for the fungus to invade our plants. Additionally, the leaf damage may have upregulated the JA defense pathway, as commonly occurs with mechanical wounding, thereby suppressing levels of SA and facilitating powdery mildew infection as a result of antagonistic SA/JA cross-talk .The mock-inoculated plants in our greenhouse study showed significantly more severe powdery mildew symptoms than both the control and virus-inoculated plants  is common and well documented (Transgenic cultivars are often grown near wild populations and because gene flow between cultivated squash and wild gourds (cumented , it is lcumented . Studiescumented . In popucumented .C. pepo that could be bred into cultivated C. pepo. Moreover, recent studies have revealed that vertical transmission rates of ZYMV are 1.6% and that infected plants are virtually asymptomatic (C. pepo show the same immune response to powdery mildew infection that horizontally infected ZYMV plants show in our studies, it could serve as a low pesticide alternative to extend the growing season. Similar cross-protection strategies in which a mild strain of one virus protects against a more virulent strain of that virus or another pathogen, have been effectively used in other crops (see reviews in In addition to the implications for management of transgenic introgressives in wild populations, these data have implications for applied agriculture. Powdery mildew is a major economic pest of cultivated squashes and pumpkins worldwide by decreasing yield by as much as 50% and shortening the growing season . Moreoveptomatic . If vertCucurbita pepo pathosystem reveal that a virus resistance transgene would spread rapidly through natural populations of wild gourd when only viral disease is present in the population. However, when multiple pathogens are present in the population, we see both pre-infection interactions and post-infection interactions among pathogens that would limit the spread of the transgene. The parallels between these mechanisms and those in animal systems suggest that these tractable plant systems might provide a test-bed for general tests of pathogen–pathogen–host interactions.Plants in agricultural and especially natural populations are part of complex pathosystems consisting of a community of interacting organisms  that havhttps://scholarsphere.psu.edu/collections/4f16c298n.The data supporting this manuscript are available via Penn State’s ScholarSphere and can be found at the following URL: JH, AS, and MF conceived and designed the experiments. JH performed the experiments. AH assisted with phytohormone experiments. JH, AS, AH, and MF analyzed the data. AS and JT contributed reagents, materials, and analysis tools. JH, AS, JT, and MF wrote the manuscript.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.


A prominent tree species coexistence mechanism suggests host-specific natural enemies inhibit seedling recruitment at high conspecific density (negative conspecific density dependence). Natural-enemy-mediated conspecific density dependence affects numerous tree populations, but its strength varies substantially among species. Understanding how conspecific density dependence varies with species’ traits and influences the dynamics of whole communities remains a challenge. Using a three-year manipulative community-scale experiment in a temperate forest, we show that plant-associated fungi, and to a lesser extent insect herbivores, reduce seedling recruitment and survival at high adult conspecific density. Plant-associated fungi are primarily responsible for reducing seedling recruitment near conspecific adults in ectomycorrhizal and shade-tolerant species. Insects, in contrast, primarily inhibit seedling recruitment of shade-intolerant species near conspecific adults. Our results suggest that natural enemies drive conspecific density dependence in this temperate forest and that which natural enemies are responsible depends on the mycorrhizal association and shade tolerance of tree species. The Janzen-Connell hypothesis posits that seedlings may be less likely to establish near conspecifics due to shared natural enemies. Here, Jia et al. show that tree species traits determine whether fungal pathogens or insect herbivores inhibit seedling recruitment and survival in a temperate forest. The Janzen-Connell hypothesis4 provides one mechanism, suggesting that host-specific natural enemies are concentrated where their host plant species occur at high densities , suppressing recruitment and survival of their host plants (negative conspecific density dependence)6. Such self-inhibition by conspecifics puts rare species at an advantage and facilitates plant species coexistence and diversity. Several empirical studies have reported that seedling recruitment and survival decrease with the density of conspecific neighbors, but are relatively unaffected by heterospecifics9. There is also increasing experimental evidence that insect herbivores and, pathogenic fungi in particular, are responsible for mediating the conspecific density dependence observed in several plant populations15. Despite growing consensus that natural enemies regulate plant population dynamics, however, the extent to which this process contributes to diversity at the community scale remains poorly known (but see refs. 17).Natural enemies  have long been postulated to play crucial roles in determining the diversity of plant communities19, which is one factor complicating assessments of its contribution to plant community diversity. This variation may be correlated with plant species’ traits22, which potentially provides a basis for extrapolation to the community-scale. There is some evidence that slow-growing, shade-tolerant tree species are less affected by conspecific neighbors than shade-intolerant species23, potentially because shade-tolerance is associated with greater investment in defense or storage, and hence lower susceptibility to natural enemies25. Additionally, types of mycorrhizal association  differ in their effectiveness in protecting plant roots from antagonists27, which could translate into variation in susceptibility of hosts to natural enemies and hence density dependence. For example, a continental-scale study reported that EM seedlings respond positively to the density of conspecific trees, while AM seedlings respond negatively28. However, the relative importance of different traits in modifying the strength of natural-enemy-mediated density dependence in intact communities remains largely untested.The strength of conspecific density dependence varies considerably among species4. Nevertheless, the relationship between latitude and the strength of conspecific density dependence remains disputed33. Inferences based on non-manipulative surveys are sensitive to analytical methods, with both marked weakening of conspecific density dependence with latitude32 and no relationship reported using the same datasets33. A meta-analysis of manipulative experiments of individual plant populations reported no consistent relationship between conspecific density dependence and latitude30. The strength of conspecific density dependence varies greatly among experiments and species so community-wide experiments have proved useful tools in determining the importance of density dependence for diversity in tropical forests17. However, community-scale experimental manipulations of natural enemies, equivalent to those performed in tropical forests (e.g. refs. 17), have not been attempted in less species-rich, temperate forests.The strength of conspecific density dependence may also vary among ecosystems. The Janzen-Connell hypothesis originally postulated that natural enemy-induced conspecific density dependence should be more negative in tropical than in temperate systems28, and that conspecific density dependence mediated by both plant-associated fungi and insects are greater for shade-intolerant than shade-tolerant species23. We manipulated the access of three groups of natural enemies, large herbivores (using fences), insect herbivores (using insecticide) and plant-associated fungi (using fungicide), to seedlings in 180 1 m2 quadrats  within a 20 m radius of each quadrat. We use the 20 m radius because intraspecific interactions are often undetectable beyond this distance37. Our results indicate that plant-associated fungi have a negative effect on seedling recruitment and survival at high conspecific adult density. Insect herbivores also inhibit seedling recruitment near conspecific adults, but they do not affect seedling survival. Additionally, which groups of natural enemies impact seedling recruitment near conspecific adults depends on two important tree species traits . Plant-associated fungi have a stronger negative effect on seedling recruitment for EM-associated and shade-tolerant species than AM-associated and shade-intolerant species. In contrast, seedling recruitment is more strongly inhibited by insect herbivores near conspecific adults for shade-intolerant species than shade-tolerant species.We report on a field experiment in an old-growth temperate forest in northeast China to test for conspecific density dependence at the community scale, to identify the groups of natural enemies that may be responsible and to examine whether variation in the strength of density dependence among plant species is associated with two important plant traits: shade tolerance and type of mycorrhizal association. We test two hypotheses: 1) Plant-associated fungi and insect herbivores are critical drivers of conspecific density dependence and hence seedling diversity and composition in this temperate forest, and (2) Species varying in mycorrhizal association and shade-tolerance differ in their sensitivity to enemy-mediated density dependence. Specifically, based on the literature we predict that AM trees are more sensitive to fungal-mediated conspecific density-dependence than EM trees Plant-asAcross the 180 quadrats, 3929 tree seedlings recruited from 16 species . Protection from fungi increased both recruitment and survival of seedlings when conspecific adult densities were high , but did not affect seedling survival appreciably. The effect of large vertebrates was not estimated to be large in any of the models , but fungicide did not appreciably ameliorate that effect . Additionally, survival of AM species was significantly less dependent on conspecific adult density . The effects of insecticide treatment on recruitment and survival did not differ between mycorrhizal association types  recorded were from five species of EM trees, most of which (93% of EM seedlings) were shade tolerant. The remaining seedlings belonged to 11 AM species, which included both shade-tolerant  and shade-intolerant  species. The influence of natural enemies on seedling recruitment and survival near adult conspecifics differed between EM and AM tree species. The relationship between conspecific adult density and recruitment of EM species was significantly more positive in the fungicide treatment than in either the control or insecticide treatments . Nevertheless, the interactive effect of adult conspecific density and fungicide on seedling recruitment was similar for shade tolerant and shade intolerant species (i.e. the three-way interaction was non-significant). Insecticide, on the other hand, increased recruitment of shade-intolerant seedlings near conspecific adults significantly more than recruitment of shade tolerant species . Shade tolerance did not influence the response of seedling survival to either conspecific adult density, biocides or their interactions  accounted for 65% of all seedlings. Recruitment of shade tolerant species increased with adult conspecific density, and this positive relationship strengthened significantly when fungicide was applied . Spraying fungicide, however, did influence species composition, significantly decreasing the dissimilarity between seedlings and surrounding adult trees  was unaffected by suppressing natural enemies , we found no evidence that attack by either plant-associated fungi or insect herbivores is related to the density of conspecific seeds and seedlings, or that it increase seedling diversity. Instead, our results suggest that density of adult conspecifics is a better predictor of natural enemy impacts on recruitment and survival of seedlings. These natural enemy impacts appear to be important, even though the net effect of conspecific density is only weakly negative in plots when natural enemies are unmanipulated. Both recruitment and survival go from negatively to positively related to conspecific adult density when fungi and, to a lesser extent, insects are excluded. As a result, seedling communities are less similar to adults than when fungi are suppressed. Several field and shade-house experiments involving small numbers of tree species have indicated that performance is inhibited near conspecific adults as a result of plant-associated fungi39 and insect herbivores40. Our results extend the experimental evidence that seedling performance is suppressed by adult conspecifics to a community scale.While the evidence that natural enemies reduce seedling performance at high conspecific density in our system is in line with previous pesticide application experiments in field conditions in tropical forests21. Our analyses identified that recruitment of EM-associated and shade-tolerant species is more strongly inhibited by adult conspecifics, although the correlation between mycorrhizal association and shade-tolerance (most EM seedlings are shade tolerant), hinders any definite distinction between the effects of the two traits. Suppression of plant-associated fungi increase seedling recruitment of EM species in areas of high conspecific adult density. While there is little difference between EM and AM species in the effects of adults on seedling recruitment, fungi do not appear to be responsible for conspecific density dependence in AM species. Interestingly, previous studies have suggested that EM seedlings should preferentially recruit near conspecific adults42, in contrast to our results. One possible explanation for this disagreement is that we concentrated on the earliest stages in the trees’ lives, before mycorrhizal associations are generally established. At this stage, the mycorrhizal colonization rate is much lower , our results suggest that the causes of density dependence may be consistent across latitudes. Several studies in tropical forests have suggested that while insects influence seedling recruitment and might mediate density dependence in some species40, plant-associated fungi play a bigger role in mediating conspecific density dependence at the community scale17. Similarly, although insects contribute to conspecific density dependence in our experiment, suppressing fungi has a greater effect on seedling recruitment and survival than insecticide application at the community scale. While fences are effective at excluding large herbivores such as wild boars and roe deer  may limit our ability to detect impacts of large herbivores on plant communities. Further, better replicated experiments that exclude large herbivores may be necessary to determine their role in shaping plant communities in these ecosystems. Although further work is necessary to determine the identity and host-specificity of insect herbivores and plant-associated fungi, perhaps using molecular techniques (e.g. refs. 58), on the basis of the evidence presented here, insects and plant-associated fungi appear to play a greater role in mediating conspecific density dependence than large herbivores.Although much of the evidence that natural enemies cause conspecific density dependence in tree seedlings has come from tropical forests17 suggests that causes of conspecific density dependence (e.g. plant-associated fungi and insect herbivores) may be consistent across latitudes. Additionally, our results indicate that the strength of density dependence largely depends on the association between the types of natural enemies and the mycorrhizal association type or shade tolerance of tree species. Although natural-enemy-mediated density dependence does not translate into increased seedling diversity, our results suggest that plant-associated fungi play a critical role in shaping species composition and turnover in our temperate forest. Notably, our study highlights the need to consider multiple types of natural enemies and traits of plant species to improve our understanding of the mechanisms of conspecific density dependence and species coexistence.Overall, our field experiment provides evidence of a community-wide Janzen-Connell effect in this temperate forest. Comparison of these results to those from similar experiments in tropical forests59, mostly as rain from June to September and snow otherwise, with the ground covered by snow from November to April. The vegetation is dominated by broad-leaf and mixed forest over slightly acidic (mean pH = 5.45) dark brown forest soil. Dominant tree species include Tilia amurensis, Fraxinus mandschurica, Quercus mongolica, and Pinus koraiensis.This study was conducted within the Changbai Mountain National Nature Reserve in northeast China . The climate is characterized by an annual mean temperature of 2.8 °C (−13.7–19.6 °C) and average annual precipitation of ~700 mm2 seedling quadrats on a grid, with quadrats separated by ~2 m and avoiding large roots. We randomly selected thirty of these quadrats for use in the experiment described here. These 30 quadrats were divided equally and randomly among three pesticide treatments: fungicide, insecticide, and control.We established three 55 m × 50 m blocks, separated by at least 200 m, within an old growth temperate forest in September 2014  at three locations within and outside each fence between September 2015 and September 2017. In addition, in each census we examined all quadrats for evidence of large mammals (e.g. footprints or feces).61. Insects were deterred by applying a broad spectrum, systemic insecticide, Engeo . We applied pesticides biweekly except during the winter. Pesticide concentrations followed the manufacturer’s guidelines . Control quadrats were sprayed with an equal volume of water.We initiated pesticide treatments in July 2015 and continued until October 2017. Plants were protected from true fungi and oomycetes through a combination of two fungicides, Amistar  and Ridomil Gold MZ 68 . Both fungicides have low toxicity to non-target organisms and have been reported to have little effect on mycorrhizal colonization62. We calculated recruitment as the number of new seedlings >1 cm tall in each quadrat in every census. Including tiny seedlings would allow us to indirectly measure the effects of natural enemies on seedling survival during the seed to seedling transition. We did not evaluate the effect of seed abundance on recruitment because the majority of tree species recruit from seed bank rather than seed rain, and the time germinating from seeds varies from months to years across species. In June 2017, we identified all trees (DBH > 5 cm) within each block and recorded their species identity, DBH and distance to each quadrat within 20 m.We conducted censuses of woody plants in June 2015, 2016, and 2017. All woody plants <1 cm DBH (Diameter at Breast Height) were tagged, mapped and identified to species in the 180 quadrats. In September of each year, we checked the status  of each existing seedling. We focused on survival status in the first few months after recruitment (seed-to-seedling transition), because seedlings are most vulnerable to natural enemies during this stage63. The range of the conspecific crowding index within the 20 m radius of each quadrat was from 0 to 0.42. We also calculated the conspecific (S.con) and heterospecific seedling densities (S.het) as the number of seedlings within each quadrat .Analyses of seedling recruitment and survival only considered tree seedlings recruited after pesticide applications were initiated. We used the crowding index (i.e. A.con and A.het) to represent the conspecific and heterospecific adult densities, which were calculated by summing the inverse-distance weighted basal areas of all conspecific and heterospecific adults within a 20-m radius of each quadrat64. We included pesticide treatment, exclosure treatment and conspecific adult density (i.e. A.con) and the two- and three-way interactions among them as fixed factors in the full model for recruitment. Heterospecific adult density (A.het) was included as a covariate in the recruitment model. For survival, we added covariates describing seedling neighborhoods (conspecific seedling density-S.con and heterospecific seedling density-S.het) in addition to all the covariates included in the recruitment model. We did not include seedling densities in recruitment analyses because the number of recruits were confounded with the seedling densities. Seedling height was also included as a covariate to account for the potential positive relationship between survival and seedling size65. To evaluate the contribution of two- or three-way interactions in explaining seedling recruitment and survival, the full models considering all interactions were compared with all possible reduced models from which one or more interactions were removed using likelihood ratio tests (χ2 with degree of freedom of the differences in the number of parameters66). The models assumed Poisson and binomial error distributions for recruitment and survival respectively, and we confirmed that model residuals were not overdispersed. To account for spatial and temporal autocorrelation between closely located plots and censuses in the data, we included block and census as categorical fixed effects14. We also considered quadrat and species identity as random intercepts and allowed the effect of census to vary among quadrats as a random effect. We did not include random slopes for different species because preliminary analyses indicated that allowing slopes to vary among species led to less parsimonious models (higher AIC) than intercept-only models, and the coefficients did not change qualitatively.We used generalized linear mixed-effects models (GLMM) to evaluate the effects of enemy types and neighboring plants on seedling recruitment and survival. All continuous explanatory variables were log-transformed and then standardized (i.e. by subtracting the mean and dividing by the standard deviation) to facilitate the direct comparison of the relative importance of each variableTo explore correlates of interspecific variation in covariate effects, we categorized each tree species by shade tolerance (shade-tolerant or shade-intolerant) and mycorrhizal association  accounted for 74% of all seedlings. To evaluate the influence of these two species on the community-wide and correlates of interspecific variation results, we conducted a sensitivity analysis. We randomly removed 25, 50 and 75% of individuals of T. amurensis and F. mandschurica from the community data sets  and refitted the models, repeating this procedure 999 times. We compared parameter estimates from these simulations to the models including all seedlings. Despite their numerical dominance of the seedling assemblage, our results were insensitive to removing random fractions of individuals of these two species 69 using the package “lme4”70.All the data analyses were conducted using R v 3.4.2Further information on research design is available in the Supplementary Information FilePeer Review FileReporting Summary


Facilitating beta-lactam antibiotic use in patients reporting beta-lactam allergies in acute care settings is important to individual patient outcomes and public health; however, few initiatives have targeted the Emergency Department (ED) setting.We implemented pathways for patients reporting prior penicillin and/or cephalosporin hypersensitivity as part of a hospital guideline in the ED of a large academic medical center in the United States. We described beta-lactam test doses, pathway compliance, hypersensitivity reactions (HSRs), and allergy record updating associated with ED-administered beta-lactam test doses from October 2016 to June 2018.310 beta-lactam antibiotic test doses were administered to patients with penicillin and/or cephalosporin allergy histories in the study period (average volume 15/month [standard deviation 4]). Test doses were to cephalosporins (85%), penicillins (12%), and carbapenems (4%). 219 (71%) of test doses were compliant with the pathways. Ten patients  had HSRs; five HSR patients (50%) had beta-lactams administered that were not pathway compliant. The allergy record was updated in 146 (47%) of patients, with improvement over the study period (p < 0.001).Inpatient approaches to prescribing beta-lactams in patients reporting beta-lactam allergies can be operationalized in the ED. Additional efforts are required to ensure guideline compliance and appropriate allergy documentation. Clostridioides difficile infections, adverse drug reactions, death).3Beta-lactam antibiotic allergies are commonly reported, but uncommonly reflective of clinically significant immunologic reactions.Although penicillin skin testing (PST) can be used in patients reporting beta-lactam allergies to optimize antibiotic use in acute care settings,,,,Penicillin and cephalosporin hypersensitivity pathways were designed to safely increase beta-lactam antibiotic use in hospitalized patients with histories of penicillin and/or cephalosporin allergy .8 These The inpatient penicillin and cephalosporin hypersensitivity pathways were implemented in the ED of one acute care hospital in 2016 with pharmacist-led education. Test doses were ordered by an ED healthcare provider, including medical doctors, nurse practitioners, and physician assistants. After pharmacist verification and preparation of the test dose procedure order, ED nurses administered test doses and performed the requisite observation.We identified all patients who had a history of penicillin and/or cephalosporin allergy and received a beta-lactam antibiotic test dose in the ED between October 2016 and June 2018. Electronic health record (EHR)-collected data included patient demographics, allergy history, and test dose outcomes. The test dose outcomes included HSR signs, symptoms, and treatment and allergy documentation updates. HSRs from the beta-lactam antibiotics included reactions that were potentially immunologic such as urticaria, angioedema, anaphylaxis, shortness of breath, and reactions concerning for organ specific reactions or severe cutaneous adverse reactions. Compliance with pathways was assessed retrospectively by a clinical pharmacist and allergist/immunologist. If there was not adequate documentation to support compliance with the pathways, noncompliance was assumed. We used a chi-squared test to compare frequencies of appropriate allergy documentation.We identified 310 patients (mean age 63 years [standard deviation (SD) 19 years] and 38% male) who received a beta-lactam antibiotic by test dose procedure in the ED. Test doses occurred on average 15 times per month (SD 4). Test doses were to cephalosporins , penicillins , and carbapenems .The pathways were followed in 219 (71%) cases overall. Among noncompliant cases , most  unnecessarily administered a test dose when a full-dose was appropriate  had HSRs ; 5 (50%)The allergy list was appropriately updated in 146 (47%) of patients overall, with improvement observed over the study period (from 18% in month one to 71% in month 21 p < 0.001). Allergy updating was more frequent for the patients with HSRs; 9 (90%) of HSR patients had their beta-lactam allergy appropriately added.We reviewed 310 beta-lactam antibiotic test doses performed in the ED for patients with reported allergies to penicillins or cephalosporins. Most test doses were for cephalosporins, and while HSRs were uncommon, half of the HSRs were triggered by the test dose. The pathways were followed in at least 71% of cases. Although overall allergy documentation was poor, it improved with time, potentially as the pathways became more familiar.Patients with penicillin allergies can and should be evaluated in all healthcare settings, but the ED presents unique operational challenges given that interventions must occur 24 hours a day, including weekends and holidays, when specialist access may be restricted. Further, patients in the ED have varied levels of acuity and lengths of stay; while some patients are ultimately admitted and need parenteral antibiotics, others are discharged home on oral antibiotics. For acutely infected patients, delays in antibiotic treatment can result in deleterious outcomes; one study documented that patients with a penicillin allergy history had a longer mean time to first antibiotic dose than those without a penicillin allergy history .The observed HSR frequency from the penicillin and cephalosporin pathways in the ED setting (3.2%) was similar to HSR frequency observed in our largest system analysis to date (3.8%).Although the majority of ED test doses followed the established hypersensitivity pathways, more than one-quarter were noncompliant, and half of the HSRs occurred in patients receiving a noncompliant test dose. Adherence in this study was notably lower than observed in the initial inpatient analyses of these pathways where 91% of test doses were guideline-compliant and no adverse reactions occurred from non-compliant test doses.Updating of a patient's allergy record is important to the quality and safety of patient care.We reviewed patients who received a beta-lactam test dose in the ED setting, but were unable to identify how many ED patients in this time-frame were eligible to receive beta-lactam test doses. Thus, we cannot completely judge the success of our implementation. Because this study was retrospective, we relied on EHR documentation that is prone to errors and omissions. To decrease miscategorization, many parts of the EHR were evaluated, including clinician and nurse documentation and medication administration records. While we were limited by sample size from doing comparative analyses, we anticipate that these initial data may motivate expansion of these pathways more broadly into ED settings across our healthcare system, and potentially to other institutions that have adopted similar approaches.We describe our experience using a guideline with penicillin and cephalosporin hypersensitivity pathways in the ED setting. ED test doses through the guideline were feasible and HSRs infrequent, but we found lower guideline compliance and recognized persistent allergy documentation challenges that require unique solutions given that the ED has quick patient throughput and a large and diverse provider pool. Still, pathway implementation facilitated standardized beta-lactam antibiotic use in patients with beta-lactam allergy histories in a timely and safe manner, likely improving the quality and safety of patient care.Partners Quality, Safety, and Value and the Partners Clinical Process Improvement Leadership Program (CPIP). Dr. Blumenthal received career development support from the NIH K01AI125631, the American Academy of Allergy Asthma and Immunology (AAAAI) Foundation, and the MGH Claflin Distinguished Scholar Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, AAAI Foundation, nor MGH.This work was supported by KGB and ESS report a licensed clinical decision support tool for inpatient beta-lactam allergy evaluation. The remaining authors have nothing to disclose.All authors confirm their consent for publication.This study protocol was approved by the Partners Human Research Committee protocol 2015P001873.