HIV Infection Modifies the Role of Prior Treponema pallidum Infection in the Clinical Presentation of Early Syphilis Among Adult Patients From Sexually Transmitted Infection Clinics in Peru.

BACKGROUND: We aimed to compare the clinical presentations (symptomatic vs. asymptomatic) with prior Treponema pallidum infection status (first infection vs. reinfection) among people with early syphilis. METHODS: We used data from PICASSO, a cohort study in Peru that enrolled people with active syphilis from May 2019 to August 2021. Study participants had early syphilis and a prior syphilis serological test result within the prior 12 months to determine prior T. pallidum infection status. We calculated prevalence ratios (PRs) of symptomatic clinical presentation (primary or secondary syphilis) by prior T. pallidum infection status, stratified by HIV infection status. In addition, we explored the association of prior T. pallidum infection status and lesion presentation, stratified by primary and secondary syphilis cases, using the Fisher exact test. RESULTS: We include 84 T. pallidum reinfection cases and 61 first infection cases. We found increased frequency of symptomatic clinical presentation among first-infection cases (39% vs. 20%; PR, 1.94; P = 0.014). This association was stronger among persons living without HIV infection (38% vs. 7%; adjusted PR, 6.63; P = 0.001) in comparison to those living with HIV infection (45% vs. 34%; adjusted PR, 1.38; P = 0.458). Among secondary syphilis cases, more participants from the reinfection group reported that their lesions improved 1 week after treatment (100% vs. 29%, P = 0.045) compared with those with a first infection. Among the primary syphilis cases, all participants reported that their lesions improved 1 week after treatment. CONCLUSIONS: Prior syphilis was associated with a decreased prevalence of symptomatic reinfection, especially among persons not living with HIV infection.

Neonatal mortality risk of vulnerable newborns by fine stratum of gestational age and birthweight for 230 679 live births in nine low- and middle-income countries, 2000-2017.

OBJECTIVE: To describe the mortality risks by fine strata of gestational age and birthweight among 230 679 live births in nine low- and middle-income countries (LMICs) from 2000 to 2017. DESIGN: Descriptive multi-country secondary data analysis. SETTING: Nine LMICs in sub-Saharan Africa, Southern and Eastern Asia, and Latin America. POPULATION: Liveborn infants from 15 population-based cohorts. METHODS: Subnational, population-based studies with high-quality birth outcome data were invited to join the Vulnerable Newborn Measurement Collaboration. All studies included birthweight, gestational age measured by ultrasound or last menstrual period, infant sex and neonatal survival. We defined adequate birthweight as 2500-3999 g (reference category), macrosomia as ≥4000 g, moderate low as 1500-2499 g and very low birthweight as <1500 g. We analysed fine strata classifications of preterm, term and post-term: ≥42+0 , 39+0 -41+6 (reference category), 37+0 -38+6 , 34+0 -36+6 ,34+0 -36+6 ,32+0 -33+6 , 30+0 -31+6 , 28+0 -29+6 and less than 28 weeks. MAIN OUTCOME MEASURES: Median and interquartile ranges by study for neonatal mortality rates (NMR) and relative risks (RR). We also performed meta-analysis for the relative mortality risks with 95% confidence intervals (CIs) by the fine categories, stratified by regional study setting (sub-Saharan Africa and Southern Asia) and study-level NMR (≤25 versus >25 neonatal deaths per 1000 live births). RESULTS: We found a dose-response relationship between lower gestational ages and birthweights with increasing neonatal mortality risks. The highest NMR and RR were among preterm babies born at <28 weeks (median NMR 359.2 per 1000 live births; RR 18.0, 95% CI 8.6-37.6) and very low birthweight (462.8 per 1000 live births; RR 43.4, 95% CI 29.5-63.9). We found no statistically significant neonatal mortality risk for macrosomia (RR 1.1, 95% CI 0.6-3.0) but a statistically significant risk for all preterm babies, post-term babies (RR 1.3, 95% CI 1.1-1.5) and babies born at 370 -386 weeks (RR 1.2, 95% CI 1.0-1.4). There were no statistically significant differences by region or underlying neonatal mortality. CONCLUSIONS: In addition to tracking vulnerable newborn types, monitoring finer categories of birthweight and gestational age will allow for better understanding of the predictors, interventions and health outcomes for vulnerable newborns. It is imperative that all newborns from live births and stillbirths have an accurate recorded weight and gestational age to track maternal and neonatal health and optimise prevention and care of vulnerable newborns.

Artificial Intelligence for Mohs and Dermatologic Surgery: A Systematic Review and Meta-Analysis.

BACKGROUND: Over the past decade, several studies have shown that potential of artificial intelligence (AI) in dermatology. However, there has yet to be a systematic review evaluating the usage of AI specifically within the field of Mohs micrographic surgery (MMS). OBJECTIVE: In this review, we aimed to comprehensively evaluate the current state, efficacy, and future implications of AI when applied to MMS for the treatment of nonmelanoma skin cancers (NMSC). MATERIALS AND METHODS: A systematic review and meta-analysis was conducted following PRISMA guidelines across several databases, including PubMed/MEDLINE, Embase, and Cochrane libraries. A predefined protocol was registered in PROSPERO, with literature search involving specific keywords related to AI and Mohs surgery for NMSC. RESULTS: From 23 studies evaluated, our results find that AI shows promise as a prediction tool for precisely identifying NMSC in tissue sections during MMS. Furthermore, high AUC and concordance values were also found across the various usages of AI in MMS, including margin control, surgical recommendations, similarity metrics, and in the prediction of stage and construction complexity. CONCLUSION: The findings of this review suggest promising potential for AI to enhance the accuracy and efficiency of Mohs surgery, particularly for NMSC.

Publishing on Topical Subjects in Total Joint Arthroplasty Is Associated With Increased Social Media Attention.

BACKGROUND: Social media platforms are often used for research dissemination and collaboration. Given the increased prevalence of online-only publications, understanding what drives research dissemination is important. Here, we analyzed factors associated with increased social media attention among peer-reviewed publications in total knee arthroplasty, total hip arthroplasty, and unicompartmental knee arthroplasty. METHODS: We analyzed publications about total knee arthroplasty, total hip arthroplasty, or unicompartmental knee arthroplasty from 2010 to 2022 using a national database. We analyzed a weighted count of social media mentions, using negative binomial regressions adjusting for days since publication. Publications on "hot topics" in arthroplasty were examined including navigation/robotics, COVID-19, race/ethnicity, body mass index, and reimbursement. There were 9,542 publications included, 4,216 (44%) were open access (OA), 338 (3.5%) included navigation, 32 (0.34%) discussed race/ethnicity, 20 (0.2%) discussed COVID-19, 3,840 (40%) were randomized studies, 30 (0.3%) discussed reimbursement, and 2,867 (30%) were in top-10 orthopaedic journals. RESULTS: Factors associated with higher weighted score included studies about COVID-19 (50 versus 6.0, P < .001), race/ethnicity (15.8 versus 6.0, P < .001), OA status (6.3 versus 5.8, P = .001), and randomized studies (6.5 versus 5.7, P < .001). Studies from top-10 journals had a lower score (5.8 versus 6.2, P = .025), as did studies about body mass index (3.4 versus 6.1, P = .001). Studies about navigation and reimbursement did not have significantly different scores. CONCLUSIONS: Studies on COVID-19, race/ethnicity, randomized studies, and OA publication were associated with increased social media while those in top-10 orthopaedic journals had lower scores. LEVEL OF EVIDENCE: Level IV, Prognostic Study.

The Definition of Anemia Matters When Using Preoperative Hemoglobin as a Screening Tool Prior to Total Hip and Knee Arthroplasty.

BACKGROUND: Preoperative anemia is common in patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA). Several definitions of anemia have been described, with no clear consensus on the optimal one for preoperative screening. We hypothesized that depending on the definition used preoperatively, the proportion of anemic patients identified who would require a postoperative allogeneic blood transfusion would vary significantly. METHODS: A total of 681,141 patients were identified in a national database who underwent either THA or TKA. Preoperative anemia was classified according to the World Health Organization (WHO) definition, Cleveland Clinic (CC) definition, or race, age, and sex-specific definition described by Beutler et al in 2006. The optimal preoperative (OP) hemoglobin thresholds to predict perioperative transfusions were also calculated using receiver operating characteristic curves. RESULTS: When using the WHO definition, 18% of anemic patients required a transfusion versus 14% (OP definition), 12% (CC definition), and 16% (Beutler definition). Similarly, 0.69% of anemic patients sustained a periprosthetic joint infection within 30 days using the WHO definition versus 0.59% (OP definition), 0.60% (CC definition), or 0.66% (Beutler definition). Using the WHO definition, 5.3% of patients would have sustained a major complication versus 4.5% (OP definition), 4.4% (CC definition), and 5.0% (Beutler definition). CONCLUSIONS: Variation in the definition of anemia for preoperative screening in THA and TKA results in substantial differences in discriminative ability to predict perioperative transfusions. The WHO definition identified the largest proportion of patients who ultimately received a perioperative transfusion.

Tumor resident, TRA anti-viral CDR3 chemical sequence motifs are associated with a better breast cancer outcome.

While for certain cancers, such as cervical cancer, the link to viral infections is very strong and very clear, other cancers represent a history of links to viral infections that are either co-morbidities or drive the cancer in ways that are not yet fully understood, for example the "hit and run" possibility. To further understand the connection of viral infections and the progress of breast cancer, we identified the chemical features of known anti-viral, T-cell receptor alpha chain (TRA) complementarity determining region-3 (CDR3) amino acid sequences among the CDR3s of breast cancer patient TRA recombinations and assessed the association of those features with patient outcomes. The application of this novel paradigm indicated consistent associations of tumor-derived, anti-CMV CDR3 chemical sequence motifs with better breast cancer patient outcomes but did not indicate an opportunity to establish risk stratifications for other cancer types. Interestingly, breast cancer samples with no detectable TRA recombinations represented a better outcome than samples with the non-anti-CMV CDR3s, further adding to a rapidly developing series of results allowing a distinction between positive and possibly harmful cancer immune responses.

Hidradenitis Suppurativa: Molecular Etiology, Pathophysiology, and Management-A Systematic Review.

Hidradenitis suppurativa is a chronic inflammatory skin condition that affects the hair follicles in areas of the body with apocrine glands. The condition is characterized by recurrent, painful nodules, abscesses, and draining sinuses that can lead to scarring and disfigurement. In this present study, we provide a focused evaluation of recent developments in hidradenitis suppurativa research, including novel therapeutics and promising biomarkers that may facilitate clinical diagnosis and treatment. We conducted a systematic review of controlled trials, randomized controlled trials, meta-analyses, case reports, and Cochrane Review articles in accordance with the PRISMA guidelines. The Cochrane Library, PubMed, EMBASE, and Epistemonikos databases were queried via Title/Abstract screen. Eligibility criteria included the following: (1) has a primary focus on hidradenitis suppurativa, (2) includes measurable outcomes data with robust comparators, (3) details the sample population, (4) English language, and (5) archived as full-text journal articles. A total of 42 eligible articles were selected for review. Qualitative evaluation identified numerous developments in our understanding of the disease's multiple potential etiologies, pathophysiology, and treatment options. It is important for individuals with hidradenitis suppurativa to work closely with a healthcare provider to develop a comprehensive treatment plan that addresses their individual needs and goals. To meet this objective, providers must keep current with developments in the genetic, immunological, microbiological, and environmental factors contributing to the disease's development and progression.

TRB CDR3 chemical complementarity with HBV epitopes correlates with increased hepatocellular carcinoma, disease-free survival.

The liver is a site of immune privilege, compared with the bladder and skin, for example. To study this attenuation of the immune response in the cancer setting, we compared quantities and features of adaptive immune receptor (IR) recombination reads obtained from hepatocellular carcinoma (HCC) and six other cancers. Of these cancers, HCC had the lowest numbers of IR recombination reads and was the only cancer with a greater number immunoglobulin rather than T-cell receptor recombination reads. To better understand the role of adaptive IRs obtained from the tumor microenvironment in shaping the outcome of HCC cases, we quantified the chemical complementarity between HCC tumor TRB and IGH complementarity determining region-3 (CDR3) amino acid (AA) sequences, and known hepatitis B virus (HBV) epitopes. High chemical complementarity between HCC-resident CDR3s and three HBV epitopes correlated with increased survival probabilities, for two sources of CDR3s representing different CDR3 recovery algorithms. These results suggest the potential of CDR3 AA sequences as biomarkers for HCC patient stratification and as guides for future development of therapeutics.

Carrier Screening Controls Transport in Conjugated Polymers at High Doping Concentrations.

Transport properties of doped conjugated polymers (CPs) have been widely analyzed with the Gaussian disorder model (GDM) in conjunction with hopping transport between localized states. These models reveal that even in highly doped CPs, a majority of carriers are still localized because dielectric permittivity of CPs is well below that of inorganic materials, making Coulomb interactions between carriers and dopant counterions much more pronounced. However, previous studies within the GDM did not consider the role of screening the dielectric interactions by carriers. Here we implement carrier screening in the Debye-Hückel formalism in our calculations of dopant-induced energetic disorder, which modifies the Gaussian density of states (DOS). Then we solve the Pauli master equation using Miller-Abrahams hopping rates with states from the resulting screened DOS to obtain conductivity and Seebeck coefficient across a broad range of carrier concentrations and compare them to measurements. Our results show that screening has significant impact on the shape of the DOS and consequently on carrier transport, particularly at high doping. We prove that the slope of Seebeck coefficient versus electric conductivity, which was previously thought to be universal, is impacted by screening and decreases for systems with small dopant-carrier separation, explaining our measurements. We also show that thermoelectric power factor is underestimated by a factor of ∼10 at higher doping concentrations if screening is neglected. We conclude that carrier screening plays a crucial role in curtailing dopant-induced energetic disorder, particularly at high carrier concentrations.

Neonatal mortality risk of vulnerable newborns: A descriptive analysis of subnational, population-based birth cohorts for 238 203 live births in low- and middle-income settings from 2000 to 2017.

OBJECTIVE: We aimed to understand the mortality risks of vulnerable newborns (defined as preterm and/or born weighing smaller or larger compared to a standard population), in low- and middle-income countries (LMICs). DESIGN: Descriptive multi-country, secondary analysis of individual-level study data of babies born since 2000. SETTING: Sixteen subnational, population-based studies from nine LMICs in sub-Saharan Africa, Southern and Eastern Asia, and Latin America. POPULATION: Live birth neonates. METHODS: We categorically defined five vulnerable newborn types based on size (large- or appropriate- or small-for-gestational age [LGA, AGA, SGA]), and term (T) and preterm (PT): T + LGA, T + SGA, PT + LGA, PT + AGA, and PT + SGA, with T + AGA (reference). A 10-type definition included low birthweight (LBW) and non-LBW, and a four-type definition collapsed AGA/LGA into one category. We performed imputation for missing birthweights in 13 of the studies. MAIN OUTCOME MEASURES: Median and interquartile ranges by study for the prevalence, mortality rates and relative mortality risks for the four, six and ten type classification. RESULTS: There were 238 203 live births with known neonatal status. Four of the six types had higher mortality risk: T + SGA (median relative risk [RR] 2.6, interquartile range [IQR] 2.0-2.9), PT + LGA (median RR 7.3, IQR 2.3-10.4), PT + AGA (median RR 6.0, IQR 4.4-13.2) and PT + SGA (median RR 10.4, IQR 8.6-13.9). T + SGA, PT + LGA and PT + AGA babies who were LBW, had higher risk compared with non-LBW babies. CONCLUSIONS: Small and/or preterm babies in LIMCs have a considerably increased mortality risk compared with babies born at term and larger. This classification system may advance the understanding of the social determinants and biomedical risk factors along with improved treatment that is critical for newborn health.

Predictors of inpatient admission likelihood and prolonged length of stay among cerebrovascular disease patients: A nationwide emergency department sample analysis.

PURPOSE: To examine the hospital- and patient-related factors associated with increased likelihood of inpatient admission and extended hospitalization. METHODS: We applied multivariate logistic regression to a subset of ED hospital and patient characteristics linearly extrapolated from the 2019 National Emergency Department Sample database (n=626,508). Patient characteristics with 10 or fewer ED visits after national extrapolation were not reported in the current study to maintain patient confidentiality, in accordance with the HCUP Data Use Agreement. All selected ED visits represented a primary diagnosis of CVD (ICD-10 codes 160-168). All reported hospital and patient characteristics were subject to adjustment for covariates. P-values < 0.05 were considered statistically significant. MAIN FINDINGS: Medicare beneficiaries report higher inpatient admission rates than uninsured OR 0.81 (0.73-0.91) and privately insured OR 0.86 (0.79-0.94) individuals. Black and Native-American patients were 37% and 55% more likely to be hospitalized long (>75th percentile) (OR 1.37 [1.25-1.50], OR 1.55 [1.14-2.10]). Northeast emergency departments reported an increased odds of admission compared to the Midwest OR (0.40-0.62), South OR 0.79 (0.63-0.98) and West OR 0.52 (0.39-0.69). Patients with multiple comorbidities (mCCI = 3+) were 226% more likely to have a longer stay OR 3.26 (3.09-3.45) than patients presenting with zero or few comorbidities. Level I, II, and III trauma centers report distinctly high odds of inpatient admission (OR 3.54 [2.84-4.42], OR 2.68 [2.14-3.35], OR 1.51 [1.25-1.84]). PRINCIPAL CONCLUSIONS: Likelihoods of inpatient admission and long hospital stays were observably stratified through multiple, independently acting hospital and patient characteristics. Significant associations were stratified by race/ethnicity, location, and clinical presentation, among others. Attention to the factors reported here may serve well to mitigate emergency department crowding and its sobering impact on United States healthcare systems and patients.

Comparative imaging findings among different primary betacoronaviruses.

Purpose: Coronaviruses (CoV) are single-stranded RNA viruses that transmit from animal species to humans, causing a threat to global health. We aim to summarize common imaging findings of 3 betacoronaviruses (b-CoVs) and the common clinical manifestation, to provide a better understanding of the courses of the disease. Material and methods: The Pubmed and Google Scholar databases were searched for the terms "SARS-CoV" OR "COVID-19" OR "MERS-CoV". Imaging-specific searches included keyword searches for "CT" AND "imaging". Clinical presentation-specific searches included keyword searches for "clinical" AND "manifestation" AND "cardio-vascular" OR "neurology" OR "gastrointestinal" OR "hematology". In total, 77 articles were selected for discussion in the current literature review. Results: Human b-CoVs infection presented consistent indications of ground-glass opacities (GGO), consolidation, and interlobular septal thickening. Pleural effusion was also common in all 3 b-CoVs, but it was least present in SARS-CoV-2 infection. Bilateral lung involvement was common to both MERS-CoV and SARS-CoV-2 infection. Cardiovascular, neurological, haematological, and gastrointestinal were common clinical presentations found in patients infected with b-CoVs. Conclusions: The comparison of imaging findings can be applied in clinical practice to distinguish the 3 CoV through different imaging modalities. It is crucial to understand the possible imaging findings and clinical presentations to better understand the course of the disease as well as prepare for future variants.

Update on the limited sensitivity of computed tomography relative to RT-PCR for COVID-19: a systematic review.

Purpose: The global and ongoing COVID-19 outbreak has compelled the need for timely and reliable methods of detection for SARS-CoV-2 infection. Although reverse transcription-polymerase chain reaction (RT-PCR) has been widely accepted as a reference standard for COVID-19 diagnosis, several early studies have suggested the superior sensitivity of computed tomography (CT) in identifying SARS-CoV-2 infection. In a previous systematic review, we stratified studies based on risk for bias to evaluate the true sensitivity of CT for detecting SARS-CoV-2 infection. This study revisits our prior analysis, incorporating more current data to assess the sensitivity of CT for COVID-19. Material and methods: The PubMed and Google Scholar databases were searched for relevant articles published between 1 January 2020, and 25 April 2021. Exclusion criteria included lack of specification regarding whether the study cohort was adult or paediatric, whether patients were symptomatic or asymptomatic, and not identifying the source of RT-PCR specimens. Ultimately, 62 studies were included for systematic review and were subsequently stratified by risk for bias using the QUADAS-2 quality assessment tool. Sensitivity data were extracted for random effects meta-analyses. Results: The average sensitivity for COVID-19 reported by the high-risk-of-bias studies was 68% [CI: 58, 80; range: 38-96%] for RT-PCR and 91% [CI: 87, 96; range: 47-100%] for CT. The average sensitivity reported by the low-risk-of-bias studies was 84% [CI: 0.75, 0.94; range: 70-97%] for RT-PCR and 78% [CI: 71, 0.86; range: 44-92%] for CT. Conclusions: On average, the high-risk-of bias studies underestimated the sensitivity of RT-PCR and overestimated the sensitivity of CT for COVID-19. Given the incorporation of recently published low-risk-of-bias articles, the sensitivities according to low-risk-of-bias studies for both RT-PCR and CT were higher than previously reported.

The low molecular weight protein tyrosine phosphatase promotes adipogenesis and subcutaneous adipocyte hypertrophy.

Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.

Effects of a potassium channel opener on brain injury and neurologic outcomes in an animal model of neonatal hypoxic-ischemic injury.

BACKGROUND: Hypoxia-ischemia (HI) is the most common cause of brain injury in newborns and the survivors often develop cognitive and sensorimotor disabilities that undermine the quality of life. In the current study, we examined the effectiveness of flupirtine, a potassium channel opener, shown previously in an animal model to have strong anti-neonatal-seizure efficacy, to provide neuroprotection and alleviate later-life disabilities caused by neonatal hypoxic-ischemic injury. METHODS: The rats were treated with a single dose of flupirtine for 4 days following HI induction in 7-day-old rats. The first dose of flupirtine was given after the induction of HI and during the reperfusion period. The effect of treatment was examined on acute and chronic brain injury, motor functions, and cognitive abilities. RESULTS: Flupirtine treatment significantly reduced HI-induced hippocampal and cortical tissue loss at acute time point. Furthermore, at chronic time point, flupirtine reduced contralateral hippocampal volume loss and partially reversed learning and memory impairments but failed to improve motor deficits. CONCLUSION: The flupirtine treatment regimen used in the current study significantly reduced brain injury at acute time point in an animal model of neonatal hypoxic-ischemic encephalopathy. However, these neuroprotective effects were not persistent and only modest improvement in functional outcomes were observed at chronic time points.

Diagnostic Tools for Coronavirus Disease (COVID-19): Comparing CT and RT-PCR Viral Nucleic Acid Testing.

OBJECTIVE. Multiple studies suggest CT should be a primary diagnostic tool for coronavirus disease (COVID-19) because they reported sensitivities with CT far superior to that of reverse transcriptase polymerase chain reaction (RT-PCR) testing. This review aimed to assess these reports and found chest CT to have a clinical utility that is limited, particularly for patients who show no symptoms and patients who are screened early in disease progression. CONCLUSION. CT has limited sensitivity for COVID-19 and a lower specificity than RT-PCR testing, and it carries a risk of exposing providers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Chest CT should be considered a supplemental diagnostic tool, particularly for patients who show symptoms.